Bispecific anti-CCL2 antibodies

ABSTRACT

The present invention relates to bispecific anti-CCL2 antibodies binding to two different epitopes on human CCL2, pharmaceutical compositions thereof, their manufacture, and use as medicaments for the treatment of cancers, inflammatory, autoimmune and ophthalmologic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to EP Application No. 19217665.9, filedDec. 18, 2019, the disclosure of which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 14, 2020, is named“P35823-US_Sequence_Listing_ST25.txt” and is 245,760 bytes in size.

FIELD OF THE INVENTION

The present invention relates to bispecific anti-CCL2 antibodies bindingto two different epitopes on human CCL2, pharmaceutical compositionsthereof, their manufacture, and use as medicaments for the treatment ofcancers, inflammatory, autoimmune and ophthalmologic diseases.

BACKGROUND OF THE INVENTION

The CCL2/CCR2 axis is the main mediator of immature myeloid cellrecruitment into the tumor. CCL2 is overexpressed by malignant cells andbinds to the extracellular matrix (ECM) building up a chemoattractantgradient. Once they reach the tumor, myeloid-derived suppressive cells(MDSCs) contribute to the pro-tumorigenic milieu bysecreting/up-regulating anti-inflammatory cytokines/receptors that inturn inhibit the initiation of an anti-tumor T cell response. In thisway, MDSCs may reduce or even impair the efficacy of any Tcell-activating therapy (Meyer et al, 2014). Therefore, the specificinhibition of the recruitment of these immature myeloid cells will boostthe efficacy of checkpoint inhibitors, T cell bispecific antibodies(TCBs) or other cancer immunotherapies (CITs). In addition, CCL2 hasalso been implicated in the promotion of angiogenesis, metastasis andtumor growth, suggesting that neutralizing CCL2 might contribute toseveral lines of anti-tumor intervention.

Targeting CCL2—as opposed to its receptor—will specifically inhibit theundesired CCL2-mediated effects, sparing those that might signal throughthe same receptor (CCR2) but different ligands (e.g. CCL7, CCL8, CCL13)which are involved in the recruitment of other immune cell populations,like Th1 and NK cells.

Clinically, CCL2 has been a preferred antibody-target in several studiesaiming at neutralizing its elevated levels caused by differentinflammatory diseases, such as rheumatoid arthritis (Haringman et al,Arthritis Rheum. 2006 August; 54(8):2387-92), idiopathic pulmonaryfibrosis (Raghu et al, Eur Respir J. 2015 December; 46(6):1740-50),diabetic nephropathy (Menne et al, Nephrol Dial Transplant (2017) 32:307-315) and cancer (Sandhu et al, Cancer Chemother Pharmacol. 2013April; 71(4):1041-50). However, its high synthesis rate together withthe observed high in vivo antibody-antigen dissociation constants (KD)have proven to be the main obstacles hindering the suppression of freeCCL2 by conventional antibodies at clinically viable doses (Fetterly etal, J Clin Pharmacol. 2013 October; 53(10):1020-7).

CCL2 neutralization appears to be more obviously relevant in patientswith elevated serum levels of CCL2, which has been observed in severaltypes of cancers like breast cancer (BC), ovarian cancer (OvCa),colorectal cancer (CRC), pancreatic cancer and prostate cancer. However,even patients within these indications who do not present this serologybut whose tumors are highly infiltrated with immune cells of the myeloidlineage might very well profit from this novel therapy due to the manyroles that CCL2 plays in the tumor context as mentioned above.

Igawa et al, Immunological Reviews 270 (2016) 132-151 describes theSweeping technology in which the generated antibody bears pH-dependentCDRs (for antibody-antigen dissociation within the acidic endosomes,leading to antigen degradation) and an engineered Fc moiety with anoptimized isoelectric point (pI) and enhanced binding to FcgammaRIIb(favoring the cellular uptake of immune complexes), and a moderateaffinity to the neonatal Fc receptor, to maintain an acceptablepharmacokinetic profile.

SUMMARY OF THE INVENTION

The present invention relates to bispecific anti-CCL2 antibodies bindingto two different epitopes on human CCL2, pharmaceutical compositionsthereof, their manufacture, and use as medicaments for the treatment ofcancers, inflammatory, autoimmune and ophthalmologic diseases.

-   One embodiment of the invention is a bispecific antibody comprising    a first antigen-binding site that (specifically) binds to a first    epitope on human CCL2 and a second different antigen-binding site    that (specifically) binds a second different epitope on human CCL2,    wherein bispecific antibody comprises a Fc domain of human IgG    isotype.-   One embodiment of the invention is a bispecific antibody comprising    a first antigen-binding site that (specifically) binds to a first    epitope on human CCL2 and a second different antigen-binding site    that (specifically) binds a second epitope on human CCL2, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 49, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 50, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 51;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 52; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 53, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 54; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising a (d) CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 2, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 3;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 5, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 6; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG isotype.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG isotype.-   In one embodiment the bispecific antibody comprises a constant heavy    chain domain of human IgG1 isotype.-   In one embodiment the in vivo clearance rate for human CCL2    (ml/day/kg) after administration of the bispecific antibody    comprising a constant heavy chain domain of human wild type IgG1    isotype (or the Fc domain thereof) is at least two fold higher (in    one embodiment at least 5 fold higher, in one embodiment at least 10    fold higher, in one embodiment at least 20 fold higher) compared to    the in vivo clearance rate for human CCL2 (ml/day/kg) after    administration of a bispecific antibody comprising a Fc gamma    receptor silenced constant heavy chain domain of human IgG1 isotype    (or the Fc domain thereof) comprising the mutations L234A, L235A,    P329G (Kabat EU numbering), when a pre-formed immune complex    consisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg    human CCL2 was administered at a single dose of 10 ml/kg into FcRn    transgenic mice.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second epitope on human CCL2, wherein    -   i) said first antigen-binding site binds to same epitope on CCL2        as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and    -   ii) said second antigen-binding site binds to same epitope on        CCL2 as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46.

In one embodiment the in vivo clearance rate for human CCL2 (ml/day/kg)after administration of the bispecific antibody comprising a constantheavy chain domain of human wild type IgG1 isotype (or the Fc domainthereof) is at least 15 fold higher, in particular at least 20 foldhigher, compared to the in vivo clearance rate for human CCL2(ml/day/kg) after administration of a bispecific antibody comprising aFc gamma receptor silenced constant heavy chain domain of human IgG1isotype (or the Fc domain thereof) comprising the mutations L234A,L235A, P329G (Kabat EU numbering), when a pre-formed immune complexconsisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg humanCCL2 was administered at a single dose of 10 ml/kg into FcRn transgenicmice.

-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second epitope on human CCL2, wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I or            T, (b) a CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62;        -   and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2            comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ            ID NO: 77 wherein X is D or E, and (c) a CDR-H3 comprising            the amino acid sequences GVFGFFXH of SEQ ID NO:78 wherein X            is D or E;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (e) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second epitope on human CCL2, wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I or            T, (b) a CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, (c) a CDR-H3 comprising            the amino acid sequence YDAHYGELDF of SEQ ID NO: 59, (d) a            FR-H1 comprising the amino acid sequence            QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO:63, (e) a FR-H2            comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID            NO:64, (f) a FR-H3 comprising the amino acid sequence            RVTITADESTSTAYMELSSLRSEDTAVY YCAR of SEQ ID NO:65, and (g) a            FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ            ID NO:66;        -   and        -   a VL domain comprising (h) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (i) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (j) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62, (k) a FR-L1 comprising the            amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID            NO:67, (1) a FR-L2 comprising the amino acid sequence            WYQQKPGQAPRLLIY of SEQ ID NO:68, (m) a FR-L3 comprising the            amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ            ID NO:69, and (n) a FR-L4 comprising the amino acid sequence            GQGTKVEIK of SEQ ID NO:70;        -   and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2            comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ            ID NO: 77 wherein X is D or E, (c) a CDR-H3 comprising the            amino acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D            or E, (d) a FR-H1 comprising the amino acid sequence            QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO:82, (e) a FR-H2            comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID            NO:83, (f) a FR-H3 comprising the amino acid sequence            RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO:84, and (g) a            FR-H4 comprising the amino acid sequence WGQGTTVTVSS of SEQ            ID NO:85;        -   and        -   a VL domain comprising (h) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (i) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, (j) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R, (k) a FR-L1 comprising the amino            acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO:86, (1) a            FR-L2 comprising the amino acid sequence WYQQKPGKAPKLLIH of            SEQ ID NO:87, (m) a FR-L3 comprising the amino acid sequence            GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO:88, and (n) a            FR-L4 comprising the amino acid sequence FGGGTKVEIK of SEQ            ID NO:89.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second epitope on human CCL2,    -   wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   J) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   K) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   L) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   M) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   N) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   O) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   P) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second epitope on human CCL2, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, X² is            P, and X³ is H, and (c) a CDR-H3 comprising the amino acid            sequence YDAHYGELDF of SEQ ID NO: 59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D, and (c) a CDR-H3 comprising the amino acid            sequences GVFGFFXH of SEQ ID NO:78 wherein X is D;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F and            X² is R, (e) a CDR-L2 comprising the amino acid sequence            GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the            amino acid sequence QQFXSAPYT of SEQ ID NO: 81 wherein X is            W;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, X² is            P, and X³ is H, and (c) a CDR-H3 comprising the amino acid            sequence YDAHYGELDF of SEQ ID NO: 59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D, and (c) a CDR-H3 comprising the amino acid            sequences GVFGFFXH of SEQ ID NO:78 wherein X is E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F and            X² is R, (e) a CDR-L2 comprising the amino acid sequence            GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the            amino acid sequence QQFXSAPYT of SEQ ID NO: 81 wherein X is            W;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   I) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   J) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   K) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   L) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   M) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   N) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   O) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   P) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   In one embodiment the bispecific antibody described herein    -   i) blocks binding of CCL2 to its receptor CCR2 in vitro        (reporter assay, IC₅₀=0.5 nM); and/or    -   ii) inhibits CCL2-mediated chemotaxis of myeloid cells in vitro        (IC₅₀=1.5 nM); and/or    -   iii) is cross-reactive to cynomolgus and human CCL2.-   In one embodiment the bispecific antibody described herein is not    cross-reactive to other CCL homologs in particular it shows 100 time    less binding to other CCL homologs (as e.g. CCL8) compared to the    binding to CCL2-   In one embodiment the bispecific antibody described herein binds to    the first and second epitope on human CCL2 in ion-dependent manner.-   In one embodiment the bispecific antibody described herein binds to    human CCL2 in pH dependent manner and wherein the first antigen    binding site and the second antigen binding site both bind to CCL2    with a higher affinity at neutral pH than at acidic pH.-   In one embodiment the bispecific antibody described herein binds to    human CCL2 with a 10 times higher affinity at pH 7.4, than at pH    5.8.-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L,        T307P, K326T and/or A330K (suitable for increasing affinity to        human FcgRIIb and decreasing affinity to other human FcgR);        and/or    -   iii) M428L, N434A and/or Y436T (suitable for increasing affinity        to FcRn for longer plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R, and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and/or    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iv) Q438R and/S440E (suitable for suppressing rheumatoid factor        binding).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iii) Q438R and/S440E (suitable for suppressing rheumatoid factor        binding).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering) Q311R and    P343R (suitable for increasing pI for enhancing uptake of antigen).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L234Y, P238D, T250V, V264I, T307P and/or A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and/or    -   iii) M428L, N434A and/or Y436T (suitable for increasing affinity        to FcRn for longer plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and iii) M428L, N434A and Y436T (suitable for        increasing affinity to FcRn for longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A and (suitable for increasing affinity to FcRn for        longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).-   In one embodiment the bispecific antibody described herein comprises    two IgG1 heavy chain constant domains (or the Fc domain thereof)    comprising (independently or in addition to the above described    mutations) the following mutations (EU numbering)    -   i) S354C and T366W in one of the heavy chain constant domains    -   ii) Y349C, T366S, L368A, Y407V in the other of the heavy chain        constant domains-   One embodiment of the invention is an (isolated) (monospecific)    antibody that (specifically) binds to a human CCL2, wherein the    antibody comprises    -   A) a VH domain comprising (a) a CDR-H1 comprising the amino acid        sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a        CDR-H2 comprising the amino acid sequence GX¹IX²IFX³TANYAQKFQG        of SEQ ID NO: 58 wherein X¹ is V, I, or H, X² is P or H, and X³        is H or G, and (c) a CDR-H3 comprising the amino acid sequence        YDAHYGELDF of SEQ ID NO: 59;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62;    -   or    -   B) a VH domain comprising (a) a CDR-H1 comprising the amino acid        sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the        amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77 wherein X        is D or E, and (c) a CDR-H3 comprising the amino acid sequences        GVFGFFXH of SEQ ID NO:78 wherein X is D or E; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (e) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   One embodiment of the invention is an (isolated) (monospecific)    antibody that (specifically) binds to a human CCL2, wherein the    antibody comprises    -   A) a VH domain comprising the amino acid sequence of SEQ ID        NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   B) a VH domain comprising the amino acid sequence of SEQ ID        NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   C) a VH domain comprising the amino acid sequence of SEQ ID        NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   D) a VH domain comprising the amino acid sequence of SEQ ID        NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   E) a VH domain comprising the amino acid sequence of SEQ ID        NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   F) a VH domain comprising the amino acid sequence of SEQ ID        NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   G) a VH domain comprising the amino acid sequence of SEQ ID        NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   H) a VH domain comprising the amino acid sequence of SEQ ID        NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;-   One embodiment of the invention is an isolated nucleic acid encoding    the (mono- or bispecific) antibody according to any one of the    preceding embodiments.-   One embodiment of the invention is a host cell comprising such    nucleic acid.-   One embodiment of the invention is a method of producing an antibody    comprising culturing such host cell so that the antibody is    produced.-   In one embodiment of the invention such method further comprises the    step of recovering the antibody from the host cell.-   One embodiment of the invention is a pharmaceutical formulation    comprising the bispecific antibody as described herein and a    pharmaceutically acceptable carrier.-   One embodiment of the invention is the bispecific antibody as    described herein for use as a medicament.-   One embodiment of the invention is the use of the bispecific as    described herein in the manufacture of a medicament.-   In one embodiment such medicament is for the treatment of cancer.-   In one embodiment such medicament is for the treatment of an    inflammatory or autoimmune disease.-   One embodiment of the invention is the bispecific antibody as    described herein for use in treating cancer.-   One embodiment of the invention is the bispecific antibody as    described herein for use in treating an inflammatory or autoimmune    disease.-   One embodiment of the invention is a method of treating an    individual having cancer comprising administering to the individual    an effective amount of the antibody as described herein.-   One embodiment of the invention is a method of treating an    individual having an inflammatory or autoimmune disease comprising    administering to the individual an effective amount of the antibody    as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 , FIG. 1A, FIG. 1B, and FIG. 1C: Surface plasmon resonance(Biacore®) sensorgrams showing binding of monospecific anti-CCL2antibodies (CNTO888 (=CNTO), 1A5, 1G9 and humanized 11K2 (=11k2) torecombinant CCL2 and CCL2 homologs.

FIG. 2 a : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibodyCNTO888-SG1 (wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2(hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody CNTO888-SG105 (Fcreceptor binding silenced IgG1) into FcRn transgenic mice.

FIG. 2 b : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 11K2-SG1(wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 11K2-SG105 (Fc receptor bindingsilenced IgG1) into FcRn transgenic mice.

FIG. 2 c : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibodyABN912-SG1 (wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2(hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody ABN912-SG105 (Fcreceptor binding silenced IgG1) into FcRn transgenic mice.

FIG. 2 d : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1A4-SG1(wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1A4-SG105 (Fc receptor bindingsilenced IgG1) into FcRn transgenic mice.

FIG. 2 e : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1A5-SG1(wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1A5-SG105 (Fc receptor bindingsilenced IgG1) into FcRn transgenic mice.

FIG. 2 f : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 1G9-SG1(wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20mg/kg monospecific anti-CCL2 antibody 1G9-SG105 (Fc receptor bindingsilenced IgG1) into FcRn transgenic mice.

FIG. 2 g : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg monospecific anti-CCL2 antibody 2F6-SG1(wild type IgG1) or b) dotted line: 0.1 mg/kg human CCL2 (hCCL2) andmonospecific anti-CCL2 antibody 20 mg/kg 2F6-SG105 (Fc receptor bindingsilenced IgG1) into FcRn transgenic mice.

FIG. 3 a and FIG. 3 b : Shows the time course of serum total mouse CCL2concentration (FIG. 3 a ) and antibody-time profile (FIG. 3 b ) afteri.v. injection of a) solid line: 20 mg/kg monospecific anti-CCL2antibodies 11K2-SG1 (wild type IgG1) and b) dotted line: 20 mg/kgmonospecific anti-CCL2 antibodies 11K2-SG105 (Fc receptor bindingsilenced IgG1) in mice.

FIG. 4 a : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody11K2/1G9-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b)dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecificanti-CCL2 antibody 11K2/1G9-PGLALA (Fc receptor binding silenced IgG1)into Balb/c mice.

FIG. 4 b : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibodyCNTO888/11K2-WT IgG1 (wild type IgG1 with intact Fc receptor binding)orb) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecificanti-CCL2 antibody CNTO888/11K2-PGLALA (Fc receptor binding silencedIgG1) into Balb/c mice.

FIG. 4 c : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibodyCNTO888/1G9-WT IgG1 (wild type IgG1 with intact Fc receptor binding) orb) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecificanti-CCL2 antibody 11K2/1G9-PGLALA (Fc receptor binding silenced IgG1)into Balb/c mice.

FIG. 4 d : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibodyCNTO888/1A5-WT IgG1 (wild type IgG1 with intact Fc receptor binding)orb) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecificanti-CCL2 antibody CNTO888/1A5-PGLALA (Fc receptor binding silencedIgG1) into Balb/c mice.

FIG. 4 e : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody 1A5/1G9-WTIgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line:0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody1A5/1G9-PGLALA (Fc receptor binding silenced IgG1) into Balb/c mice.

FIG. 4 f : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody11K2/2F6-WT IgG1 (wild type IgG1 with intact Fc receptor binding) or b)dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecificanti-CCL2 antibody 11K2/2F6-PGLALA (Fc receptor binding silenced IgG1)into Balb/c mice.

FIG. 4 g : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibodyABN912/11K2-WT IgG1 (wild type IgG1 with intact Fc receptor binding)orb) dotted line: 0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecificanti-CCL2 antibody ABN912/11K2-PGLALA (Fc receptor binding silencedIgG1) into Balb/c mice.

FIG. 4 h : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody 1A4/2F6-WTIgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line:0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody1A4/2F6-PGLALA (Fc receptor binding silenced IgG1) into Balb/c mice.

FIG. 4 i : Serum concentration of hCCL2 over time after i.v. injectionof pre-formed immune complex consisting of a) solid line: 0.1 mg/kghuman CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody 1A5/2F6-WTIgG1 (wild type IgG1 with intact Fc receptor binding) or b) dotted line:0.1 mg/kg human CCL2 (hCCL2) and 20 mg/kg bispecific anti-CCL2 antibody1A5/2F6-PGLALA (Fc receptor binding silenced IgG1) into Balb/c mice.

FIG. 5 a : Biacore® sensorgrams showing binding profile to monomericCCL2 at pH7.4 (black line) and pH5.8 (grey line) of the four modified111K2 and four CNTO888 variants, and the 16 bispecific anti-CCL2antibodies CKLO01 to CKLO16 resulting of the respective combinationantigen binding moieties of the four modified 111K2 and four CNTO888variants.

FIG. 5 b : Biacore® sensorgrams showing binding profile to monomericCCL2, of the four modified 11K2 and four CNTO888 variants, and the 16bispecific anti-CCL2 antibodies CKLO01 to CKLO16 resulting of therespective combination antigen binding moieties of the four modified11K2 and four CNTO888 variants. An additional dissociation phase atpH5.8 was integrated into the BIACORE® assay immediately after thedissociation phase at pH 7.4.

FIG. 6 : Biacore® sensorgrams showing binding profile of bispecificanti-CCL2 antibodies CKLO01, CKLO02, CKLO03 and CKLO04 to monomeric CCL8at pH7.4 (black line) and pH5.8 (grey line).

FIG. 7 a : Serum concentration of hCCL2 over time after injection ofpre-formed immune complex consisting of hCCL2 and bispecific anti-CCL2antibodies (parental CNTO/11K2 and pH dependent variants CKLO01, CKLO02,CKLO03 and CKLO04) into SCID mice.

FIG. 7 b : Serum concentration of hCCL2 over time after injection ofpre-formed immune complex consisting of hCCL2 and CKLO03 (with IgG1 wildtype Fc) or CKLO03-SG1099, (CKLO03 with enhanced pI Fc) into SCID mice.

FIG. 8 : Chemotaxis Assay: Bispecific anti-CCL2 antibodies withidentical CDRs and variable regions VH/VL, namely CKLO2-IgG1 wild typeand CKLO2-SG1095, but different Fc moieties, can inhibit the migrationof THP-1 cells with identical potencies (IC₅₀=0.2 μg/ml; FIG. 8 , leftpanel). Similarly, CCL2-0048, the parent unmodified bispecific antibodyCNTO888/11k2k2 IgG1 of CKLO2, which is non-pH dependent, also shows anIC₅₀ of 0.2 μg/ml, since pH-dependency is critical for antigen sweeping,a phenomenon that does not take place in this assay.

The corresponding monospecific antibodies CNTO888 IgG1 and humanized11k2 IgG1 display IC₅₀ values of 0.3 and 0.7 μg/ml, respectively, whilethe huIgG1 isotype control shows no inhibition (FIG. 8 , right panel).

FIG. 9 : In vivo anti-tumor activity in a genetically-modified mousemodel. Treatment of mouse tumor model with Mab CKLO2-IgG1 (Fc wild typeIgG1) and CKLO2-SG1099 ((=CKLO2 pI-enhanced). Tumor volumes (left),tumor weights (middle), and M-MDSC infiltrate (right) at end of study.(vehicle in black, CKLO2 wild type IgG1 in grey, and CKLO2pI-enhanced Fc(CKLO2-SG1099) in white bars/dotted line)

FIG. 10 : Serum total (left) and free (right) CCL2 levels during the invivo anti-tumor activity study (see efficacy in FIG. 9 ) under treatmentwith bispecific anti-CCL2 antibodies (vehicle in black, CKLO2 wild typeIgG1 in grey, and pI-enhanced Fc (CKLO2-SG1099) in white bars/dottedline).

FIG. 11 : Proof of concept study of CCL2 sweeping efficiency incynomolgus monkeys. Total antibody concentration-time profiles in serumof cynomolgus monkeys; left panel: average concentration-time profilesof the four antibodies is presented over seven days; Group 1:monospecific CNTO888-SG1 (=IgG1 wild type) anti-CCL2 antibody (n=3animals) as control of maximal total CCL2 accumulation; group 2: abiparatopic anti-CCL2 antibody CKLO2-SG1 (IgG1 wild type) with pHdependent target binding but no Fc-modifications (n=3); group 3: abiparatopic anti-CCL2 antibody CKLO2-SG1100 with pH dependent targetbinding and Fc-pI and further modifications (n=4) and group 4:biparatopic anti-CCL2 antibody CKLO2-SG1095 with pH dependent targetbinding, Fc-pI and FcγRIIb affinity enhanced and further modifications(n=4); right panel: individual concentration-time profile of individual4 (group 2) is presented over the duration of the PK study (70 days).

FIG. 12 : Proof of concept study of CCL2 sweeping efficiency incynomolgus monkeys. Total CCL2 concentration-time profiles in serum ofcynomolgus monkeys; left panel: average total CCL2 concentration-timeprofiles of the four antibodies is presented over seven days; rightpanel: individual total CCL2 concentration-time profile of individual 4(group 2) is presented over the duration of the PK study (70 days).

FIG. 13 : Free CCL2 concentration-time profiles in serum of cynomolgusmonkeys; left panel: average free CCL2 concentration-time profiles ofthe four antibodies is presented over seven days; right panel:individual free CCL2 concentration-time profile of individual 4 (group2) is presented over the duration of the PK study (70 days); averageprofiles were calculated using a value of 0.01 ng/mL (lower limit ofquantification) for samples that were below detection limit.

FIG. 14 : PK/PD study of CCL2 sweeping efficiency in cynomolgus monkeys.Total CKL02-SG1095 concentration-time profiles in serum of cynomolgusmonkeys (CKL02-SG1095 treatment with different concentrations (group1-3)); left panel: average concentration-time profiles (n=4) for thethree dose levels are presented over seven days; right panel: individualconcentration-time profiles of two ADA-negative individual animals (25mg/kg dose group) are presented over the duration of the study (98days).

FIG. 15 : PK/PD study of CCL2 sweeping efficiency in cynomolgus monkeys.Total CCL2 concentration-time profiles in serum of cynomolgus monkeysunder CKL02-SG1095 treatment with different concentrations (group 1-3)and in comparison with CNTO888-SG1 treatment (group 4); left panel:average total CCL2 concentration-time profiles (error bars indicate SD)of the four study groups is presented over seven days; right panel:individual total CCL2 concentration-time profiles of ADA-negativeanimals from groups 3 (n=2, error bars indicate range) and 4 (n=3, errorbars indicate SD) are presented over the duration of the PK study (98days).

FIG. 16 : PK/PD study of CCL2 sweeping efficiency in cynomolgus monkeys.Free CCL2 concentration-time profiles in serum of cynomolgus monkeys;left panel: average free CCL2 concentration-time profiles (error barsindicate SD) of the four study groups is presented over seven days(CKL02-SG1095 treatment with different concentrations (group 1-3) and incomparison with CNTO888-SG1 treatment (group 4)); right panel: averagefree CCL2 concentration-time profiles of ADA-negative animals fromgroups 3 (n=2, error bars indicate range) and 4 (n=3, error barsindicate SD) are presented over the duration of the PK study (70 days);average profiles were calculated using a value of 0.01 ng/mL (lowerlimit of quantification) for samples that were below detection limit.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to bispecific anti-CCL2 antibodies bindingto two different epitopes on human CCL2, pharmaceutical compositionsthereof, their manufacture, and use as medicaments for the treatment ofcancers, inflammatory, autoimmune and ophthalmologic diseases. So theantibody comprises a first antigen-binding site that (specifically)binds to a first epitope on human CC2 and a second differentantigen-binding site that (specifically) binds a different secondepitope.

The present invention includes bispecific antibodies comprising a firstantigen-binding site that (specifically) binds to a first epitope onhuman CCL2 and a second antigen-binding site that (specifically) binds asecond epitope on human CCL2,

-   -   wherein    -   i) said first antigen-binding site binds to same epitope on CCL2        as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and    -   ii) said second antigen-binding site binds to same epitope on        CCL2 as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46.

In one embodiment the in vivo clearance rate for human CCL2 (ml/day/kg)after administration of the bispecific antibody comprising a constantheavy chain domain of human wild type IgG1 isotype (or the Fc domainthereof) is at least 15 fold higher, in particular at least 20 foldhigher, compared to the in vivo clearance rate for human CCL2(ml/day/kg) after administration of a bispecific antibody comprising aFc gamma receptor silenced constant heavy chain domain of human IgG1isotype (or the Fc domain thereof) comprising the mutations L234A,L235A, P329G (Kabat EU numbering), when a pre-formed immune complexconsisting of 20 mg/kg of each bispecific antibody and 0.1 mg/kg humanCCL2 was administered at a single dose of 10 ml/kg into FcRn transgenicmice.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an antibody. In certain embodiments, epitopedeterminant includes chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. The present inventionrelates to bispecific anti-CCL2 antibodies binding to two differentepitopes on human CCL2. The terms first and second epitope refer to twodifferent epitopes on humans CCL2. So the second epitope is differentfrom the first epitope. One can easily determine whether an antibodybinds to the same epitope as, or competes for binding with, a referenceanti-CCL2 antigen binding site by using routine methods known in theart. For example, to determine if a test antibody binds to the sameepitope as a reference anti-CCL2 antigen binding site of the invention,the reference antibody is allowed to bind to CCL2 domain thereof undersaturating conditions. Next, the ability of a test antibody to bind tohuman CCL2 is assessed. If the test antibody is able to bind to humanCCL2 following saturation binding with the reference anti-CCL2 antigenbinding site, it can be concluded that the test antibody binds to adifferent epitope than the reference anti-CCL2 antigen binding site. Onthe other hand, if the test antibody is not able to bind to human CCL2following saturation binding with the reference anti-CCL2 antibody, thenthe test antibody may bind to the same epitope as the epitope bound bythe reference anti-CCL2 antibody of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference antibody or if steric blocking (or another phenomenon) isresponsible for the lack of observed binding. Experiments of this sortcan be performed using ELISA, RIA, surface plasmon resonance (e.g.Biacore), flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art. In accordance with certainembodiments of the present invention, two antibodies bind to the same(or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excessof one antibody inhibits binding of the other by at least 50% butpreferably 75%, 90% or even 99% as measured in a competitive bindingassay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).

Alternatively, two antibodies are deemed to bind to the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies are deemed to have “overlapping epitopes” if onlya subset of the amino acid mutations that reduce or eliminate binding ofone antibody reduce or eliminate binding of the other.

To determine if an antibody competes for binding with a referenceanti-CCL2 antibody, the above-described binding methodology is performedin two orientations: In a first orientation, the reference antibody isallowed to bind to CCL2 under saturating conditions followed byassessment of binding of the test antibody to human CCL2. In a secondorientation, the test antibody is allowed to bind to an CCL2 moleculeunder saturating conditions followed by assessment of binding of thereference antibody to humans CCL2. If, in both orientations, only thefirst (saturating) antibody is capable of binding to the CCL2 molecule,then it is concluded that the test antibody and the reference antibodycompete for binding to CCL2. As will be appreciated by a person ofordinary skill in the art, an antibody that competes for binding with areference antibody may not necessarily bind to the same epitope as thereference antibody, but may sterically block binding of the referenceantibody by binding an overlapping or adjacent epitope.

When used herein, the term “CCL2”, “human CCL2”, which also called“MCP-1” is meant the 76 amino acid sequence referenced in NCBI recordaccession No. NP_002973 and variously known as CCL2, MCP-1 (monocytechemotactic protein 1), SMC-CF (smooth muscle cell chemotactic factor),LDCF (lymphocyte-derived chemotactic factor), GDCF (glioma-derivedmonocyte chemotactic factor), TDCF (tumor-derived chemotactic factors),HC11 (human cytokine 11), MCAF (monocyte chemotactic and activatingfactor). The gene symbol is SCYA2, the JE gene on human chromosome 17,and the new designation is CCL2 (Zlotnik, Yoshie 2000. Immunity 12:121-127). JE is the mouse homolog of human MCP-1/CCL2.

Handel and others (Biochemistry. 1996; 35:6569-6584) determined thesolution structure of a CCL2 dimer. These studies indicated that thesecondary structure of CCL2 consists of four β-sheets. Additionally, theresidues responsible for the dimerization interface of CCL2 weredescribed by Zhang and Rollins (Mol Cell Biol. 1995; 15:4851-4855). Theprotein complex appears elongated with the two monomers oriented in sucha way that they form a large pocket. Structures of monomeric and dimericCCL2 in two crystal forms, the so-called I and P forms, have also beendetermined (Lubkowski et al., Nat Struct Biol. 1997; 4:64-69). Paoliniet al, (J Immunol. 1994 Sep. 15; 153(6):2704-17), described thatMCP1/CCL2 exists as a monomer at physiologically relevantconcentrations: By analysing rec. CCL2 protein (purchased fromPeprotech) with size exclusion HPLC, sedimentation equilibriumultracentrifugation and chemical cross-linking, they could show that theweight fraction of monomeric and dimeric forms of MCP-1 depends on theconcertation in vitro. Finally, Seo and colleagues (J Am Chem Soc. 2013Mar. 20; 135(11):4325-32) could show by ion mobility mass spectrometrythe presence of injected CCL2 in both monomeric and dimeric forms underphysiological conditions.

Thus “wild type CCL-2” (wt CCL2) can exist as monomer but actually canalso form dimers at physiological concentrations. This monomer-dimerequilibrium is certainly different and has to be carefully taken intoaccount for all in vitro experiments described where differentconcentrations might be used. To avoid any uncertainties, we generatedpoint mutated CCL2 variants: The “P8A” variant of CCL2 carries amutation in the dimerization interface resulting in an inability to forma dimer leading to a defined, pure CCL2 monomer. In contrast, the “T10C”variant of CCL2 results in a fixed dimer of CCL2 (J Am Chem Soc. 2013Mar. 20; 135(11):4325-32).

The CCL2/CCR2 axis is the main mediator of immature myeloid cellrecruitment into the tumor. CCL2 is overexpressed by malignant cells andbinds to the extracellular matrix (ECM) building up a chemoattractantgradient. Once they reach the tumor, myeloid-derived suppressive cells(MDSCs) contribute to the pro-tumorigenic milieu bysecreting/up-regulating anti-inflammatory cytokines/receptors that inturn inhibit the initiation of an anti-tumor T cell response. In thisway, MDSCs may reduce or even impair the efficacy of any Tcell-activating therapy (Meyer et al, 2014). Therefore, the specificinhibition of the recruitment of these immature myeloid cells will boostthe efficacy of checkpoint inhibitors, T cell bispecific and cancerimmune therapies. In addition, CCL2 has also been implicated in thepromotion of angiogenesis, metastasis and tumor growth, suggesting thatneutralizing CCL2 might contribute to several lines of anti-tumorintervention.

Targeting CCL2—as opposed to its receptor—will specifically inhibit theundesired CCL2-mediated effects, sparing those that might signal throughthe same receptor (CCR2) but different ligands (e.g. CCL7, CCL8, CCL13)which are involved in the recruitment of other immune cell populations,like Th1 and NK cells.

Clinically, CCL2 has been a preferred antibody-target in several studiesaiming at neutralizing its elevated levels caused by differentinflammatory diseases, such as rheumatoid arthritis (Haringman et al,2006), idiopathic pulmonary fibrosis (Raghu et al, 2015), diabeticnephropathy (Menne et al, 2017) and cancer (Sandhu et al, 2013).However, its high synthesis rate together with the observed high in vivoantibody-antigen dissociation constants (KD) have proven to be the mainobstacles hindering the suppression of free CCL2 by conventionalantibodies at clinically viable doses (Fetterly et al, 2013).

CCL2 neutralization appears to be more obviously relevant in patientswith elevated serum levels of CCL2, which has been observed in severaltypes of cancers like breast cancer (BC), ovarian cancer (OvCa),colorectal cancer (CRC), pancreatic cancer and prostate cancer. However,even patients within these indications who do not present this serologybut whose tumors are highly infiltrated with immune cells of the myeloidlineage might very well profit from this novel therapy due to the manyroles that CCL2 plays in the tumor context as mentioned above.

As used herein, an antibody “binding to human CCL2”, “specificallybinding to human CCL2”, “that binds to human CCL2” or “anti-CCL2antibody” refers to an antibody specifically binding to the human CCL2antigen with a binding affinity of a K_(D)-value of 5.0×10⁻⁸ mol/l orlower, in one embodiment of a K_(D)-value of 1.0×10⁻⁹ mol/l or lower, inone embodiment of a K_(D)-value of 5.0×10⁻⁸ mol/l to 1.0×10⁻¹³ mol/l.

The binding affinity is determined with a standard binding assay, suchas surface plasmon resonance technique (BIAcore®, GE-Healthcare Uppsala,Sweden) e.g. using constructs comprising CCL2 extracellular domain (e.g.in its natural occurring 3 dimensional structure). In one embodimentbinding affinity is determined with a standard binding assay usingexemplary soluble CCL2.

Antibody specificity refers to selective recognition of the antibody fora particular epitope of an antigen. Natural antibodies, for example, aremonospecific.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen.

The term “bispecific antibody that binds to (human) CCL2”, “biparatopicantibody that binds to (human) CCL2”, “bispecific anti-CCL2 antibody”,“biparatopic anti-CCL2 antibody” as used herein means that the antibodyis able to specifically bind to at least two different epitopes on(human) CCL2. Typically, such bispecific antibody comprises twodifferent antigen binding sites (two different paratopes), each of whichis specific for a different epitope of (human) CCL2. In certainembodiments the bispecific antibody is capable of binding two differentand non-overlapping epitopes on CCL2, which means that the two differentantigen binding sites do not compete for binding to CCL2.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antibody. As such, the term“monovalent binding to an antigen” denotes the presence of one (and notmore than one) antigen binding site specific for the antigen in theantibody.

The terms “antigen binding site” refers to the site or region, i.e. oneor several amino acid residues, of an antibody which providesinteraction with the antigen. For example, the antigen binding site ofan antibody comprises amino acid residues from the complementaritydetermining regions (CDRs). In one embodiment the antigen binding siteof an antibody comprises the comprises amino acid residues from the VHand VL. A native immunoglobulin molecule typically has two antigenbinding sites; a Fab molecule typically has a single antigen bindingsite. “Antigen binding moiety” refers to a polypeptide moleculecomprising an antigen binding site that specifically binds to anantigenic determinant. Antigen binding moieties include antibodies andfragments thereof as further defined herein. Particular antigen bindingmoieties include an antigen binding domain of an antibody, comprising anantibody heavy chain variable region and an antibody light chainvariable region. In certain embodiments, the antigen binding moietiesmay comprise antibody constant regions as further defined herein andknown in the art. Useful heavy chain constant regions include any of thefive isotypes: α, δ, ε, γ, or μ. Useful light chain constant regionsinclude any of the two isotypes: κ and λ.

As used herein, the term “antigenic determinant” or “antigen” refers toa site on a polypeptide macromolecule to which an antigen bindingmoiety/site binds, forming an antigen binding moiety-antigen complex.Useful antigenic determinants can be found, for example, on the surfacesof tumor cells, on the surfaces of virus-infected cells, on the surfacesof other diseased cells, on the surface of immune cells, free in bloodserum, and/or in the extracellular matrix (ECM).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively. Preferably the bispecific antibodies of theinvention are of human IgG isotype, more preferably of humans IgG1isotype. The terms IgG isotype and IgG1 isotype as used herein refer tothe human IgG isotype and human IgG1 isotype. Typically the differentIgG isotypes exist in the form of slightly different allotypes based onallelic variation among the IgG subclasses (see Vidarsson et al.; FrontImmunol 5 (2014) Article 520, 1-17). An “effective amount” of an agent,e.g., a pharmaceutical formulation, refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore, an antibody produced by a host cell byexpression of a specific nucleic acid molecule encoding a full-lengthheavy chain may include the full-length heavy chain, or it may include acleaved variant of the full-length heavy chain (also referred to hereinas a “cleaved variant heavy chain”). This may be the case where thefinal two C-terminal amino acids of the heavy chain are glycine (G446)and lysine (K447, numbering according to Kabat EU index). Therefore, theC-terminal lysine (Lys447), or the C-terminal glycine (Gly446) andlysine (K447), of the Fc region may or may not be present. Amino acidsequences of heavy chains including Fc domains (or a subunit of an Fcdomain as defined herein) are denoted herein without C-terminalglycine-lysine dipeptide if not indicated otherwise. In one embodimentof the invention, a heavy chain including a subunit of an Fc domain asspecified herein, comprised in an antibody or bispecific antibodyaccording to the invention, comprises an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment of the invention, a heavy chain including asubunit of an Fc domain as specified herein, comprised in an antibody orbispecific antibody according to the invention, comprises an additionalC-terminal glycine residue (G446, numbering according to EU index ofKabat). Compositions of the invention, such as the pharmaceuticalcompositions described herein, comprise a population of antibodies orbispecific antibodies of the invention. The population of antibodies orbispecific antibodies may comprise molecules having a full-length heavychain and molecules having a cleaved variant heavy chain. The populationof antibodies or bispecific antibodies may consist of a mixture ofmolecules having a full-length heavy chain and molecules having acleaved variant heavy chain, wherein at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% of the antibodies or bispecificantibodies have a cleaved variant heavy chain. In one embodiment of theinvention a composition comprising a population of antibodies orbispecific antibodies of the invention comprises an antibody orbispecific antibody comprising a heavy chain including a subunit of anFc domain as specified herein with an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). In one embodiment of the invention a composition comprising apopulation of antibodies or bispecific antibodies of the inventioncomprises an antibody or bispecific antibody comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine residue (G446, numbering according to EUindex of Kabat). In one embodiment of the invention such a compositioncomprises a population of antibodies or bispecific antibodies comprisedof molecules comprising a heavy chain including a subunit of an Fcdomain as specified herein; molecules comprising a heavy chain includinga subunit of a Fc domain as specified herein with an additionalC-terminal glycine residue (G446, numbering according to EU index ofKabat); and molecules comprising a heavy chain including a subunit of anFc domain as specified herein with an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to EU indexof Kabat). Unless otherwise specified herein, numbering of amino acidresidues in the Fc region or constant region is “according to the EUnumbering system”, also called “numbering according to the EU index ofKabat” or “Kabat EU numbering”, as described in Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991 (see also above). A“subunit” of an Fc domain as used herein refers to one of the twopolypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the CDR and FR sequences generally appear in the followingsequence in VH (or VL):FR-HT(L1)-CDR-H1(L1)-FR-H2(L2)-CDR-H2(L2)-FR-H3(L3)-CDR-H3(L3)-FR-H4(L4).

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat, E. A. et al., Sequences of Proteins of Immunological Interest,5th ed., Bethesda Md. (1991), NIH Publication 91-3242, Vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human CDRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human antibody, and all or substantially all of the FRs correspondto those of a human antibody. A humanized antibody optionally maycomprise at least a portion of an antibody constant region derived froma human antibody. A “humanized form” of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization.

The term “complementarity determining regions” or “CDRs” as used hereinrefers to each of the regions of an antibody variable domain which arehypervariable in sequence and/or form structurally defined loops(“hypervariable loops”) and/or contain the antigen-contacting residues(“antigen contacts”). Generally, antibodies comprise six CDRs: three inthe VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2,CDR-L3). Exemplary CDRs herein include:

-   (a) hypervariable loops occurring at amino acid residues 26-32    (CDR-L1), 50-52 (CDR-L2), 91-96 (CDR-L3), 26-32 (CDR-H1), 53-55    (CDR-H2), and 96-101 (CDR-H3) (Chothia and Lesk, J. Mol. Biol.    196:901-917 (1987));-   (b) CDRs occurring at amino acid residues 24-34 (CDR-L1), 50-56    (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and    95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of    Immunological Interest, 5th Ed. Public Health Service, National    Institutes of Health, Bethesda, Md. (1991));-   (c) antigen contacts occurring at amino acid residues 27c-36    (CDR-L1), 46-55 (CDR-L2), 89-96 (CDR-L3), 30-35b (CDR-H1), 47-58    (CDR-H2), and 93-101 (CDR-H3) (MacCallum et al. J. Mol. Biol. 262:    732-745 (1996)); and-   (d) combinations of (a), (b), and/or (c), including CDR amino acid    residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (vL3), 31-35    (CDR-H1), 50-63 (CDR-H2), and 95-102 (CDR-H3).

Unless otherwise indicated, CDR-residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity see,e.g., Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding a mono- or bispecific anti-CCL2antibody” refers to one or more nucleic acid molecules encoding antibodyheavy and light chains (or fragments thereof), including such nucleicacid molecule(s) in a single vector or separate vectors, and suchnucleic acid molecule(s) present at one or more locations in a hostcell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (CDRs). (See, e.g., Kindt,T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See e.g., Portolano, S. et al., J. Immunol.150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

I. Compositions and Methods

In one aspect, the invention is based, in part, on the finding that thebispecific antibodies as described herein use different anti-CCL2antigen binding sites as first and second antigen binding site/moiety.These anti-CCL2 antibodies bind to certain epitopes of CCL2 with highspecificity, and have ability to specifically inhibit binding of CCL2 toits receptor CCR2. They show improved immune complex formation comparedto monospecific antibodies and improved CCL2 abrogation in vivo.

Bispecific-Anti-CCL2 Antibodies

Bispecific Antibodies

Bispecific antibodies as described herein are monoclonal antibodies thathave binding specificities for at least two different epitopes on CCL2.

Techniques for making multi- and bispecific antibodies include, but arenot limited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see,e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26(1997)). Multi-specific antibodies may also be made by engineeringelectrostatic steering effects for making antibody Fc-heterodimericmolecules (see, e.g., WO 2009/089004); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992) and WO 2011/034605); using the common lightchain technology for circumventing the light chain mis-pairing problem(see, e.g., WO 98/50431); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. JImmunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites,including for example, “Octopus antibodies,” or DVD-Ig are also includedherein (see, e.g. WO 2001/77342 and WO 2008/024715). Other examples ofmultispecific antibodies with three or more antigen binding sites can befound in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO2010/145792,and WO 2013/026831. The bispecific antibody or antigen binding fragmentthereof also includes a “Dual Acting FAb” or “DAF” comprising an antigenbinding site that binds to CCL2 as well as another different antigen, ortwo different epitopes of CCL2 (see, e.g., US 2008/0069820 and WO2015/095539).

Multi-specific antibodies may also be provided in an asymmetric formwith a domain crossover in one or more binding arms of the same antigenspecificity, i.e. by exchanging the VH/VL domains (see e.g., WO2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, andKlein at al., MAbs 8 (2016) 1010-20), also called CrossMabs.Asymmetrical binding arms can also be engineered by introducing chargedor non-charged amino acid mutations into domain interfaces to directcorrect Fab pairing. See e.g., WO 2016/172485.

Various further molecular formats for multispecific antibodies are knownin the art and are included herein (see e.g., Spiess et al., Mol Immunol67 (2015) 95-106).

Preferred Bispecific Antibody Formats.

According to particular embodiments of the invention, the bispecificantibody described herein with a domain crossover in one or more bindingarms of the same antigen specificity, i.e. by exchanging the VH/VLdomains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CLdomains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g.,WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108(2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20).

Charge modifications in such bispecific antibodies especially those withan exchange of the VH/VL domains (see WO 2015/150447): The bispecificantibodies of the invention may comprise amino acid substitutions in Fabmolecules comprised therein which are particularly efficient in reducingmispairing of light chains with non-matching heavy chains(Bence-Jones-type side products), which can occur in the production ofFab-based bi-/antibodies with a VH/VL exchange in one (or more, in caseof molecules comprising more than two antigen-binding Fab molecules) oftheir binding arms (see also PCT publication no. WO 2015/150447,particularly the examples therein, incorporated herein by reference inits entirety). The ratio of a desired bispecific antibody compared toundesired side products, in particular Bence Jones-type side productsoccurring in bispecific antibodies with a VH/VL domain exchange in oneof their binding arms, can be improved by the introduction of chargedamino acids with opposite charges at specific amino acid positions inthe CH1 and CL domains (sometimes referred to herein as “chargemodifications”).

Accordingly, in some embodiments wherein the first and the secondantigen binding moiety of the bispecific antibody are both Fabmolecules, and in one of the antigen binding moieties (particularly thesecond antigen binding moiety) the variable domains VL and VH of the Fablight chain and the Fab heavy chain are replaced by each other,

i) in the constant domain CL of the first antigen binding moiety theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the first antigen binding moiety the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index); orii) in the constant domain CL of the second antigen binding moiety theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the second antigen binding moiety the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index).

The bispecific antibody does not comprise both modifications mentionedunder i) and ii). The constant domains CL and CH1 of the antigen bindingmoiety having the VH/VL exchange are not replaced by each other (i.e.remain unexchanged).

In a more specific embodiment,

i) in the constant domain CL of the first antigen binding moiety theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding moiety the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); orii) in the constant domain CL of the second antigen binding moiety theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In one such embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the firstantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the firstantigen binding moiety the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) (numbering according to Kabat), and inthe constant domain CH1 of the first antigen binding moiety the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thefirst antigen binding moiety the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the first antigen bindingmoiety the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, if amino acid substitutions according to theabove embodiments are made in the constant domain CL and the constantdomain CH1 of the first antigen binding moiety, the constant domain CLof the first antigen binding moiety is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second antigen binding moiety instead of in theconstant domain CL and the constant domain CH1 of the first antigenbinding moiety. In particular, such embodiments, the constant domain CLof the second antigen binding moiety is of kappa isotype.

Accordingly, in one embodiment, in the constant domain CL of the secondantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 or the amino acidat position 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In still another embodiment, in the constant domain CL of the secondantigen binding moiety the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the secondantigen binding moiety the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In another embodiment, in the constant domain CL of the second antigenbinding moiety the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by arginine (R) (numbering according to Kabat), and in theconstant domain CH1 of the second antigen binding moiety the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

To improve heterodimerization of the Fc domain of these asymmetric(heterodimeric) proteins, in one embodiment according to these aspectsof the invention, in the first subunit of the Fc domain the threonineresidue at position 366 is replaced with a tryptophan residue (T366W),and in the second subunit of the Fc domain the tyrosine residue atposition 407 is replaced with a valine residue (Y407V) and optionallythe threonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (numberings according to Kabat EU index).

In a further embodiment according to these aspects of the invention, inthe first subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C) or the glutamicacid residue at position 356 is replaced with a cysteine residue (E356C)(particularly the serine residue at position 354 is replaced with acysteine residue), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C) (numberings according to Kabat EU index).

Alternative heterodimerization techniques are described below under “Fcdomains” and are also contemplated as further embodiments of theinvention.

In still a further embodiment according to these aspects of theinvention, the Fc domain is a human IgG₁ Fc domain.

Fc Domains and Modifications

In particular embodiments, the bispecific antibody of the inventioncomprises an Fc domain composed of a first and a second subunit. It isunderstood, that the features of the Fc domain described herein inrelation to the bispecific antibody can equally apply to an Fc domaincomprised in an antibody of the invention.

The Fe domain of the bispecific antibody consists of a pair ofpolypeptide chains comprising heavy chain domains of an immunoglobulinmolecule. For example, the Fc domain of an immunoglobulin G (IgG)molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgGheavy chain constant domains. The two subunits of the Fc domain arecapable of stable association with each other. In one embodiment, thebispecific antibody of the invention comprises not more than one Fcdomain.

In one embodiment, the Fc domain of the bispecific antibody is an IgG Fcdomain. In a particular embodiment, the Fc domain is an IgG₁ Fc domain.In another embodiment the Fc domain is an IgG₄ Fc domain. In a morespecific embodiment, the Fc domain is an IgG₄ Fc domain comprising anamino acid substitution at position S228 (Kabat EU numbering),particularly the amino acid substitution S228P. This amino acidsubstitution reduces in vivo Fab arm exchange of IgG₄ antibodies (seeStubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).In a further particular embodiment, the Fc domain is a human Fc domain.In an even more particular embodiment, the Fc domain is a human IgG₁ Fcdomain.

The Fc domains of IgG isotype are characterized bay various propertiesbased e.g. on their interaction with the Fc gamma Receptors or with theneonatal Fc receptor (FcRn) (see e.g. see Vidarsson et al.; FrontImmunol 5 (2014) Article 520, 1-17).

Fc Domain Modifications Promoting Heterodimerization

Bispecific antibodies according to the invention comprise differentantigen binding moieties, which may be fused to one or the other of thetwo subunits of the Fc domain, thus the two subunits of the Fc domainare typically comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of bispecific antibodiesin recombinant production, it will thus be advantageous to introduce inthe Fc domain of the bispecific antibody a modification promoting theassociation of the desired polypeptides.

Accordingly, in particular embodiments, the Fc domain of the bispecificantibody according to the invention comprises a modification promotingthe association of the first and the second subunit of the Fc domain.The site of most extensive protein-protein interaction between the twosubunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.Thus, in one embodiment said modification is in the CH3 domain of the Fcdomain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomodimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (e.g. VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the bispecific antibody which reduce heavy/light chainmispairing and Bence Jones-type side products.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain of the bispecific antibody an amino acidresidue is replaced with an amino acid residue having a larger sidechain volume, thereby generating a protuberance within the CH3 domain ofthe first subunit which is positionable in a cavity within the CH3domain of the second subunit, and in the CH3 domain of the secondsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a smaller side chain volume, thereby generating acavity within the CH3 domain of the second subunit within which theprotuberance within the CH3 domain of the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in (the CH3 domain of) the first subunit ofthe Fc domain (the “knobs” subunit) the threonine residue at position366 is replaced with a tryptophan residue (T366W), and in (the CH3domain of) the second subunit of the Fc domain (the “hole” subunit) thetyrosine residue at position 407 is replaced with a valine residue(Y407V). In one embodiment, in the second subunit of the Fc domainadditionally the threonine residue at position 366 is replaced with aserine residue (T366S) and the leucine residue at position 368 isreplaced with an alanine residue (L368A) (numberings according to KabatEU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C) (particularly the serineresidue at position 354 is replaced with a cysteine residue), and in thesecond subunit of the Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) (numberingsaccording to Kabat EU index). Introduction of these two cysteineresidues results in formation of a disulfide bridge between the twosubunits of the Fc domain, further stabilizing the dimer (Carter, JImmunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesthe amino acid substitutions S354C and T366W, and the second subunit ofthe Fc domain comprises the amino acid substitutions Y349C, T366S, L368Aand Y407V (numbering according to Kabat EU index).

In a particular embodiment the antigen binding moiety that binds to thesecond antigen is fused (optionally via the first antigen bindingmoiety, which binds to CCL2, and/or a peptide linker) to the firstsubunit of the Fc domain (comprising the “knob” modification). Withoutwishing to be bound by theory, fusion of the antigen binding moiety thatbinds a second antigen, such as an activating T cell antigen, to theknob-containing subunit of the Fc domain will (further) minimize thegeneration of antibodies comprising two antigen binding moieties thatbind to an activating T cell antigen (steric clash of twoknob-containing polypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment, the heterodimerization approach described in EP1870459, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment for the bispecificantibody of the invention are amino acid mutations R409D; K370E in oneof the two CH3 domains (of the Fc domain) and amino acid mutationsD399K; E357K in the other one of the CH3 domains of the Fc domain(numbering according to Kabat EU index).

In another embodiment, the bispecific antibody of the inventioncomprises amino acid mutation T366W in the CH3 domain of the firstsubunit of the Fc domain and amino acid mutations T366S, L368A, Y407V inthe CH3 domain of the second subunit of the Fe domain, and additionallyamino acid mutations R409D; K370E in the CH3 domain of the first subunitof the Fc domain and amino acid mutations D399K; E357K in the CH3 domainof the second subunit of the Fc domain (numberings according to Kabat EUindex).

In another embodiment, the bispecific antibody of the inventioncomprises amino acid mutations S354C, T366W in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations Y349C, T366S,L368A, Y407V in the CH3 domain of the second subunit of the Fc domain,or said bispecific antibody comprises amino acid mutations Y349C, T366Win the CH3 domain of the first subunit of the Fc domain and amino acidmutations S354C, T366S, L368A, Y407V in the CH3 domains of the secondsubunit of the Fc domain and additionally amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain and aminoacid mutations D399K; E357K in the CH3 domain of the second subunit ofthe Fc domain (all numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment, a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment, the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment, the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F4051, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V,K409F. In a further embodiment, a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment, the second CH3 domain furthercomprises amino acid mutations K392E, T411E, D399R and S400R (numberingsaccording to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment, a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment, the bispecific antibody or its Fc domain is of IgG₂subclass and the heterodimerization approach described in WO 2010/129304is used alternatively.

In an alternative embodiment, a modification promoting association ofthe first and the second subunit of the Fc domain comprises amodification mediating electrostatic steering effects, e.g. as describedin PCT publication WO 2009/089004. Generally, this method involvesreplacement of one or more amino acid residues at the interface of thetwo Fc domain subunits by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment, a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment, the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment, the heterodimerization approach describedin WO 2007/147901 is used alternatively. In one embodiment, a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment, the heterodimerization approach describedin WO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

The term “wild type (WT) IgG or IgG1” as used herein for the bispecificanti-CCL2 antibodies refers to a bispecific antibody which comprises anIgG or IgG1 constant heavy chain which may comprise the above describedmodifications/mutations promoting heterodimerization but which does notcomprise further Fc domain modifications/mutations increasing orreducing Fc receptor binding and/or effector function as describedbelow.

Fc Domain Modifications/Mutations Increasing or Reducing Fc ReceptorBinding and/or Effector Function:

Modification of the Bispecific Anti-CCL2 Antibodies Via SweepingTechnology

The bispecific anti-CCL2 antibodies were modified using the sweepingtechnology to enable the bispecific anti-CCL2 antibodies to abrogatefree CCl2 over longer time periods to enable sustained a biologicaleffect like anti-cancer efficacy in vivo.

The Sweeping concept is described e.g. in Igawa et al, ImmunologicalReviews 270 (2016) 132-151, WO2012/122011, WO2016/098357, andWO2013/081143 which are incorporated herein by reference.

The present invention provides methods for facilitating antibodymediated antigen uptake into cells, by reducing the antigen-bindingactivity (binding ability) in the acidic pH range of the above-describedantibody to less than its antigen-binding activity in the neutral pHrange; and this facilitates antigen uptake into cells. The presentinvention also provides methods for facilitating antibody-mediatedantigen uptake into cells, which are based on altering at least oneamino acid in the antigen-binding domain of the above-described antibodywhich facilitates antigen uptake into cells. The present invention alsoprovides methods for facilitating antibody-mediated antigen uptake intocells, which are based on substituting histidine for at least one aminoacid or inserting at least one histidine into the antigen-binding domainof the above-described antibody which facilitates antigen uptake intocells.

Herein, “antigen uptake into cells” mediated by an antibody means thatantigens are taken up into cells by endocytosis. Meanwhile, herein,“facilitate the uptake into cells” means that the rate of intracellularuptake of antibody bound to an antigen in plasma is enhanced, and/or thequantity of recycling of uptaken antigen to the plasma is reduced. Thismeans that the rate of uptake into cells is facilitated as compared tothe antibody before increasing the human FcRn-binding activity of theantibody in the neutral pH range, or before increasing the humanFcRn-binding activity and reducing the antigen-binding activity (bindingability) of the antibody in the acidic pH range to less than itsantigen-binding activity in the neutral pH range. The rate is improvedpreferably as compared to intact human IgG, and more preferably ascompared to intact human IgG. Thus, in the present invention, whetherantigen uptake into cells is facilitated by an antibody can be assessedbased on an increase in the rate of antigen uptake into cells. The rateof antigen uptake into cells can be calculated, for example, bymonitoring over time reduction in the antigen concentration in theculture medium containing human FcRn-expressing cells after adding theantigen and antibody to the medium, or monitoring over time the amountof antigen uptake into human FcRn-expressing cells. Using methods of thepresent invention for facilitating the rate of antibody-mediated antigenuptake into cells, for example, the rate of antigen elimination from theplasma can be enhanced by administering antibodies. Thus, whetherantibody-mediated antigen uptake into cells is facilitated can also beassessed, for example, by testing whether the rate of antigenelimination from the plasma is accelerated or whether the total antigenconcentration in plasma is reduced by administering an antibody.

Herein, “total antigen concentration in plasma” means the sum ofantibody bound antigen and non-bound antigen concentration, or “freeantigen concentration in plasma” which is antibody non-bound antigenconcentration. Various methods to measure “total antigen concentrationin plasma” or “free antigen concentration in plasma” is well known inthe art as described hereinafter.

“Intact human IgG” (or “wild type (WT) human IgG) as used herein ismeant an unmodified (except with respect to the potential modificationsfor heterodimerization above) human IgG and is not limited to a specificclass of IgG. This means that human IgG1, IgG2, IgG3 or IgG4 can be usedas “intact human IgG” as long as it can bind to the human FcRn in theacidic pH range. Preferably, “intact human IgG” can be human IgG1.

The present invention also provides methods for increasing the number ofantigens to which a single antibody can bind. More specifically, thepresent invention provides methods for increasing the number of antigensto which a single antibody having human FcRn-binding activity in theacidic pH range can bind, by increasing the human FcRn-binding activityof the antibody in the neutral pH range. The present invention alsoprovides methods for increasing the number of antigens to which a singleantibody having human FcRn-binding activity in the acidic pH range canbind, by altering at least one amino acid in the human FcRn-bindingdomain of the antibody.

The present invention provides methods for facilitatingantibody-mediated antigen uptake into cells. More specifically, thepresent invention provides methods for facilitating the antigen uptakeinto cells by an antibody having human FcRn-binding activity in theacidic pH range, which are based on increasing the human FcRn-bindingactivity of the antibody in the neutral pH range. The present inventionalso provides methods for improving antigen uptake into cells by anantibody having human FcRn-binding activity in the acidic pH range,which are based on altering at least one amino acid in the humanFcRn-binding domain of the antibody.

The present invention also provides methods for facilitating antigenuptake into cells by an antibody having human FcRn-binding activity inthe acidic pH range, which are based on using a human FcRn-bindingdomain comprising an amino acid sequence with a substitution of adifferent amino acid for at least one amino acid selected from those ofpositions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265,270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315,317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424,428, 433, 434, and 436 (EU numbering) in the parent IgG Fc domain of thehuman FcRn-binding domain comprising the Fc domain of parent IgG.

The present invention also provides methods for facilitatingantibody-mediated antigen uptake into cells, by reducing theantigen-binding activity (binding ability) in the acidic pH range of theabove-described antibody to less than its antigen-binding activity inthe neutral pH range; and this facilitates antigen uptake into cells.The present invention also provides methods for facilitatingantibody-mediated antigen uptake into cells, which are based on alteringat least one amino acid in the antigen-binding domain of theabove-described antibody which facilitates antigen uptake into cells.The present invention also provides methods for facilitatingantibody-mediated antigen uptake into cells, which are based onsubstituting histidine for at least one amino acid or inserting at leastone histidine into the antigen-binding domain of the above-describedantibody which facilitates antigen uptake into cells.

Herein, “antigen uptake into cells” mediated by an antibody means thatantigens are taken up into cells by endocytosis. Meanwhile, herein,“facilitate the uptake into cells” means that the rate of intracellularuptake of antibody bound to an antigen in plasma is enhanced, and/or thequantity of recycling of uptaken antigen to the plasma is reduced. Thismeans that the rate of uptake into cells is facilitated as compared tothe antibody before increasing the human FcRn-binding activity of theantibody in the neutral pH range, or before increasing the humanFcRn-binding activity and reducing the antigen-binding activity (bindingability) of the antibody in the acidic pH range to less than itsantigen-binding activity in the neutral pH range. The rate is improvedpreferably as compared to intact human IgG, and more preferably ascompared to intact human IgG. Thus, in the present invention, whetherantigen uptake into cells is facilitated by an antibody can be assessedbased on an increase in the rate of antigen uptake into cells. The rateof antigen uptake into cells can be calculated, for example, bymonitoring over time reduction in the antigen concentration in theculture medium containing human FcRn-expressing cells after adding theantigen and antibody to the medium, or monitoring over time the amountof antigen uptake into human FcRn-expressing cells. Using methods of thepresent invention for facilitating the rate of antibody-mediated antigenuptake into cells, for example, the rate of antigen elimination from theplasma can be enhanced by administering antibodies. Thus, whetherantibody-mediated antigen uptake into cells is facilitated can also beassessed, for example, by testing whether the rate of antigenelimination from the plasma is accelerated or whether the total antigenconcentration in plasma is reduced by administering an antibody.

Herein, “total antigen concentration in plasma” means the sum ofantibody bound antigen and non-bound antigen concentration, or “freeantigen concentration in plasma” which is antibody non-bound antigenconcentration. Various methods to measure “total antigen concentrationin plasma” or “free antigen concentration in plasma” is well known inthe art as described hereinafter.

“Intact human IgG” (or “wild type IgG”) as used herein is meant anunmodified human IgG ((except with respect to the potentialmodifications for heterodimerization above) and is not limited to aspecific class of IgG. This means that human IgG1, IgG2, IgG3 or IgG4can be used as “intact human IgG” as long as it can bind to the humanFcRn in the acidic pH range. Preferably, “intact human IgG” can be humanIgG1.

“Parent IgG” as used herein means an unmodified IgG that is subsequentlymodified to generate a variant as long as a modified variant of parentIgG can bind to human FcRn in the acidic pH range (therefore, parent IgGdoes not necessary requires binding activity to human FcRn in the acidiccondition). The parent IgG may be a naturally occurring IgG, or avariant or engineered version of a naturally occurring IgG. Parent IgGmay refer to the polypeptide itself, compositions that comprise theparent IgG, or the amino acid sequence that encodes it. It should benoted that “parent IgG” includes known commercial, recombinantlyproduced IgG as outlined below. The origin of “parent IgG” is notlimited and may be obtained from any organisms of non-human animals orhuman. Preferably, organism is selected from mouse, rat, guinea pig,hamster, gerbil, cat, rabbit, dog, goat, sheep, cow, horse, camel, andnon-human primate. In another embodiment, “parent IgG” can also beobtained from cynomolgus, marmoset, rhesus, chimpanzee or human.Preferably, “parent IgG” is obtained from human IgG1 but not limited toa specific class of IgG. This means that human IgG1, IgG2, IgG3, or IgG4can be appropriately used as “parent IgG”. In the similar manner, anyclass or subclass of IgGs from any organisms hereinbefore can bepreferably used as “parent IgG”. Example of variant or engineeredversion of a naturally occurring IgG is described in Curr OpinBiotechnol. 2009 December; 20(6): 685-91, Curr Opin Immunol. 2008August; 20(4): 460-70, Protein Eng Des Sel. 2010 April; 23(4): 195-202,WO 2009/086320, WO 2008/092117, WO 2007/041635 and WO 2006/105338, butnot limited thereto.

The present invention also provides methods for increasing the abilityto eliminate plasma antigen by administering antibodies. In the presentinvention, “methods for increasing the ability to eliminate plasmaantigen” is synonymous to “methods for augmenting the ability of anantibody to eliminate antigen from plasma”. More specifically, thepresent invention provides methods for increasing the ability toeliminate plasma antigen by an antibody having human FcRn-bindingactivity in the acidic pH range, by increasing the human FcRn-bindingactivity of the antibody in the neutral pH range. The present inventionalso provides methods for increasing the ability to eliminate plasmaantigen by an antibody having human FcRn-binding activity in the acidicpH range, which are based on altering at least one amino acid in thehuman FcRn-binding domain of the antibody.

The present invention also provides methods for increasing the abilityto eliminate plasma antigen by an antibody having human FcRn-bindingactivity in the acidic pH range, by using a human FcRn-binding domaincomprising an amino acid sequence with a substitution of at least oneamino acid selected from those of positions 237, 238, 239, 248, 250,252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305,307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380,382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering)in the parent IgG Fc domain of the human FcRn-binding domain comprisingthe Fc domain of parent IgG with a different amino acid.

The present invention also provides methods for increasing the abilityto eliminate plasma antigen by an antibody, by reducing theantigen-binding activity in the acidic pH range of the above-describedantibody with improved ability to eliminate plasma antigen as comparedto the antigen-binding activity in the neutral pH range. The presentinvention also provides methods for increasing the ability to eliminateplasma antigen by an antibody, by altering at least one amino acid inthe antigen-binding domain of the above-described antibody with improvedability to eliminate plasma antigen. The present invention also providesmethods for increasing the ability to eliminate plasma antigen byadministering an antibody, by substituting histidine for at least oneamino acid or inserting at least one histidine into the antigen-bindingdomain of the above-described antibody with improved ability toeliminate plasma antigen.

Herein, the “ability to eliminate plasma antigen” means the ability toeliminate antigen from the plasma when antibodies are administered orsecreted in vivo. Thus, “increase in the ability of antibody toeliminate plasma antigen” herein means that the rate of antigenelimination from the plasma is accelerated upon administration of theantibody as compared to before increasing the human FcRn-bindingactivity of the antibody in the neutral pH range or before increasingthe human FcRn-binding activity and simultaneously reducing itsantigen-binding activity in the acidic pH range to less than that in theneutral pH range. Increase in the activity of an antibody to eliminateantigen from the plasma can be assessed, for example, by administering asoluble antigen and an antibody in vivo, and measuring the concentrationof the soluble antigen in plasma after administration. When theconcentration of soluble antigen in plasma after administration of thesoluble antigen and antibody is reduced by increasing the humanFcRn-binding activity of the antibody in the neutral pH range, or byincreasing its human FcRn-binding activity and simultaneously reducingits antigen-binding activity in the acidic pH range to less than that inthe neutral pH range, the ability of antibody to eliminate plasmaantigen can be judged to be increased. A form of soluble antigen can beantibody bound antigen or antibody non-bound antigen whose concentrationcan be determined as “antibody bound antigen concentration in plasma”and “antibody non-bound antigen concentration in plasma” respectively(The latter is synonymous to “free antigen concentration in plasma”.Since “total antigen concentration in plasma” means the sum of antibodybound antigen and non-bound antigen concentration, or “free antigenconcentration in plasma” which is antibody non-bound antigenconcentration, the concentration of soluble antigen can be determined as“total antigen concentration in plasma”. Various methods for measuring“total antigen concentration in plasma” or “free antigen concentrationin plasma” are well known in the art as described hereinafter.

The present invention also provides methods for improving thepharmacokinetics of antibodies. More specifically, the present inventionprovides methods for improving the pharmacokinetics of the antibodyhaving human FcRn-binding activity in the acidic pH range by increasingthe human FcRn-binding activity of the antibody in the neutral pH range.Furthermore, the present invention provides methods for improving thepharmacokinetics of an antibody having human FcRn-binding activity inthe acidic pH range by altering at least one amino acid in the humanFcRn-binding domain of the antibody.

The present invention also provides methods for improving thepharmacokinetics of an antibody having human FcRn-binding activity inthe acidic pH range by using a human FcRn-binding domain comprising anamino acid sequence with a substitution of different amino acid for atleast one amino acid selected from those of positions 237, 238, 239,248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298,303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360,376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EUnumbering) in the parent IgG Fc domain of the human FcRn-binding domaincomprising the Fc domain of IgG.

The plasma concentration of free antigen not bound to the antibody orthe ratio of free antigen concentration to the total concentration canbe determined by methods known to those skilled in the art, for example,by the method described in Pharm Res. 2006 January; 23 (1): 95-103.Alternatively, when an antigen exhibits a particular function in vivo,whether the antigen is bound to an antibody that neutralizes the antigenfunction (antagonistic molecule) can be assessed by testing whether theantigen function is neutralized. Whether the antigen function isneutralized can be assessed by assaying an in vivo marker that reflectsthe antigen function. Whether the antigen is bound to an antibody thatactivates the antigen function (agonistic molecule) can be assessed byassaying an in vivo marker that reflects the antigen function.

Determination of the plasma concentration of free antigen and ratio ofthe amount of free antigen in plasma to the amount of total antigen inplasma, in vivo marker assay, and such measurements are not particularlylimited; however, the assays are preferably carried out after a certainperiod of time has passed after administration of the antibody. In thepresent invention, the period after administration of the antibody isnot particularly limited; those skilled in the art can determine theappropriate period depending on the properties and the like of theadministered antibody. Such periods include, for example, one day afteradministration of the antibody, three days after administration of theantibody, seven days after administration of the antibody, 14 days afteradministration of the antibody, and 28 days after administration of theantibody. Herein, “plasma antigen concentration” means either “totalantigen concentration in plasma” which is the sum of antibody boundantigen and non-bound antigen concentration or “free antigenconcentration in plasma” which is antibody non-bound antigenconcentration.

Total antigen concentration in plasma can be lowered by administrationof antibody of the present invention by 2-fold, 5-fold, 10-fold,20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or evenhigher compared to the administration of a reference antibody comprisingthe intact human IgG Fc domain as a human FcRn-binding domain orcompared to when antigen-binding domain molecule of the presentinvention is not administered.

In another aspect, the invention provides bispecific anti-CCL2antibodies that exhibit pH-dependent binding characteristics. As usedherein, the expression “pH-dependent binding” means that the antibodyexhibits “reduced binding to CCL2 at acidic pH as compared to itsbinding at neutral pH” (for purposes of the present disclosure, bothexpressions may be used interchangeably). For example, antibodies “withpH-dependent binding characteristics” include antibodies that bind toCCL2 with higher affinity at neutral pH than at acidic pH. In certainembodiments, the bispecific antibodies of the present invention bind toCCL2 with at least 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more timeshigher affinity at neutral pH than at acidic pH. In some embodiments,the antibodies bind to CCL2 with higher affinity at pH7.4 than at pH5.8.In further embodiments, the antibodies bind to CCL2 with at least 2, 3,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 200, 400, 1000, 10000, or more times higher affinity at pH7.4than at pH5.8.

When an antigen is a soluble protein, the binding of an antibody to theantigen can result in an extended half-life of the antigen in plasma(i.e., reduced clearance of the antigen from plasma), since the antibodycan have a longer half-life in plasma than the antigen itself and mayserve as a carrier for the antigen. This is due to the recycling of theantigen-antibody complex by FcRn through the endosomal pathway in cell(Roopenian, Nat. Rev. Immunol. 7(9): 715-725 (2007)). However, anantibody with pH-dependent binding characteristics, which binds to itsantigen in neutral extracellular environment while releasing the antigeninto acidic endosomal compartments following its entry into cells, isexpected to have superior properties in terms of antigen neutralizationand clearance relative to its counterpart that binds in a pH-independentmanner (Igawa et al., Nature Biotechnol. 28(11):1203-1207 (2010);Devanaboyina et al., mAbs 5(6):851-859 (2013); WO 2009/125825).

The “affinity” of an antibody for CCL2, for purposes of the presentdisclosure, is expressed in terms of the KD of the antibody. The KD ofan antibody refers to the equilibrium dissociation constant of anantibody-antigen interaction. The greater the KD value is for anantibody binding to its antigen, the weaker its binding affinity is forthat particular antigen. Accordingly, as used herein, the expression“higher affinity at neutral pH than at acidic pH” (or the equivalentexpression “pH-dependent binding”) means that the KD of the antibodybinding to CCL2 at acidic pH is greater than the KD of the antibodybinding to CCL2 at neutral pH. For example, in the context of thepresent invention, an antibody is considered to bind to CCL2 with higheraffinity at neutral pH than at acidic pH if the KD of the antibodybinding to CCL2 at acidic pH is at least 2 times greater than the KD ofthe antibody binding to CCL2 at neutral pH. Thus, the present inventionincludes antibodies that bind to CCL2 at acidic pH with a KD that is atleast 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more times greater thanthe KD of the antibody binding to CCL2 at neutral pH. In anotherembodiment, the KD value of the antibody at neutral pH can be 10-7 M,10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less. In anotherembodiment, the KD value of the antibody at acidic pH can be 10-9 M,10-8 M, 10-7 M, 10-6 M, or greater.

In further embodiments an antibody is considered to bind to with ahigher affinity at neutral pH than at acidic pH if the KD of theantibody binding to CCL2 at pH5.8 is at least 2 times greater than theKD of the antibody binding to CCL2 at pH7.4. In some embodiments theprovided antibodies bind to CCL2 at pH5.8 with a KD that is at least 3,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 200, 400, 1000, 10000, or more times greater than the KD of theantibody binding to CCL2 at pH7.4. In another embodiment, the KD valueof the antibody at pH7.4 can be 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11M, 10-12 M, or less. In another embodiment, the KD value of the antibodyat pH5.8 can be 10-9 M, 10-8 M, 10-7 M, 10-6 M, or greater.

The binding properties of an antibody for a particular antigen may alsobe expressed in terms of the kd of the antibody. The kd of an antibodyrefers to the dissociation rate constant of the antibody with respect toa particular antigen and is expressed in terms of reciprocal seconds(i.e., sec-1). An increase in kd value signifies weaker binding of anantibody to its antigen. The present invention therefore includesantibodies that bind to CCL2 with a higher kd value at acidic pH than atneutral pH. The present invention includes antibodies that bind to CCL2at acidic pH with a kd that is at least 2, 3, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000,10000, or more times greater than the kd of the antibody binding to CCL2at neutral pH. In another embodiment, the kd value of the antibody atneutral pH can be 10-2 l/s, 10-3 l/s, 10-4 l/s, 10-5 l/s, 10-6 l/s, orless. In another embodiment, the kd value of the antibody at acidic pHcan be 10-3 l/s, 10-2 l/s, 10-1 l/s, or greater. The invention alsoincludes antibodies that bind to CCL2 with a higher kd value at pH5.8than at pH7.4. The invention includes antibodies that bind to CCL2 atpH5.8 with a kd that is at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, ormore times greater than the kd of the antibody binding to CCL2 at pH7.4.In another embodiment, the kd value of the antibody at pH7.4 can be 10-2l/s, 10-3 l/s, 10-4 l/s, 10-5 l/s, 10-6 l/s, or less. In anotherembodiment, the kd value of the antibody at pH5.8 can be 10-3 l/s, 10-2l/s, 10-1 l/s, or greater.

In certain instances, a “reduced binding to CCL2 at acidic pH ascompared to its binding at neutral pH” is expressed in terms of theratio of the KD value of the antibody binding to CCL2 at acidic pH tothe KD value of the antibody binding to CCL2 at neutral pH (or viceversa). For example, an antibody may be regarded as exhibiting “reducedbinding to CCL2 at acidic pH as compared to its binding at neutral pH”,for purposes of the present invention, if the antibody exhibits anacidic/neutral KD ratio of 2 or greater. In certain embodiments, thepH5.8/pH7.4 KD ratio for an anti-CCL2 antibody of the present inventionis 2 or greater. In certain exemplary embodiments, the acidic/neutral KDratio for an antibody of the present invention can be 2, 3, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 400, 1000, 10000, or greater. In another embodiment, the KD valueof the antibody at neutral pH can be 10-7 M, 10-8 M, 10-9 M, 10-10 M,10-11 M, 10-12 M, or less. In another embodiment, the KD value of theantibody at acidic pH can be 10-9 M, 10-8 M, 10-7 M, 10-6 M, or greater.In further instances an antibody may be regarded as exhibiting “reducedbinding to CCL2 at acidic pH as compared to its binding at neutral pH”,if the antibody exhibits an pH5.8/pH7.4 KD ratio of 2 or greater. Incertain exemplary embodiments, the pH5.8/pH7.4 KD ratio for the antibodycan be 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 200, 400, 1000, 10000, or greater. In anotherembodiment, the KD value of the antibody at pH7.4 can be 10-7 M, 10-8 M,10-9 M, 10-10 M, 10-11 M, 10-12 M, or less. In another embodiment, theKD value of the antibody at pH5.8 can be 10-9 M, 10-8 M, 10-7 M, 10-6 M,or greater.

In certain instances, a “reduced binding to CCL2 at acidic pH ascompared to its binding at neutral pH” is expressed in terms of theratio of the kd value of the antibody binding to CCL2 at acidic pH tothe kd value of the antibody binding to CCL2 at neutral pH (or viceversa). For example, an antibody may be regarded as exhibiting “reducedbinding to CCL2 at acidic pH as compared to its binding at neutral pH”,for purposes of the present invention, if the antibody exhibits anacidic/neutral kd ratio of 2 or greater. In certain exemplaryembodiments, the pH5.8/pH7.4 kd ratio for an antibody of the presentinvention is 2 or greater. In certain exemplary embodiments, theacidic/neutral kd ratio for an antibody of the present invention can be2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 400, 1000, 10000, or greater. In another embodiment,the kd value of the antibody at neutral pH can be 10-2 l/s, 10-3 l/s,10-4 l/s, 10-5 l/s, 10-6 l/s, or less. In another embodiment, the kdvalue of the antibody at acidic pH can be 10-3 l/s, 10-2 l/s, 10-1 l/s,or greater. In certain exemplary embodiments, the pH5.8/pH7.4 kd ratiofor an antibody of the present invention can be 2, 3, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400,1000, 10000, or greater. In another embodiment, the kd value of theantibody at pH7.4 can be 10-2 l/s, 10-3 l/s, 10-4 l/s, 10-5 l/s, 10-6l/s, or less. In another embodiment, the kd value of the antibody atpH5.8 can be 10-3 l/s, 10-2 l/s, 10-1 l/s, or greater.

As used herein, the expression “acidic pH” means a pH of 4.0 to 6.5. Theexpression “acidic pH” includes pH values of any one of 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In particular aspects,the “acidic pH” is 5.8.

As used herein, the expression “neutral pH” means a pH of 6.7 to about10.0. The expression “neutral pH” includes pH values of any one of 6.7,6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, and 10.0. In particular aspects, the “neutral pH” is7.4.

KD values, and kd values, as expressed herein, may be determined using asurface plasmon resonance-based biosensor to characterizeantibody-antigen interactions. KD values, and kd values can bedetermined at 25 degrees C. or 37 degrees C.

In a further aspect, the invention provides a bispecific anti-CCL2antibody that forms an immune complex (i.e. antigen-antibody complex)with CCL2. In certain embodiments, two or more bispecific anti-CCL2antibodies bind to two or more CCL2 molecules to form an immune complex.This is possible because CCL2 exists as a homodimer containing two CCL2molecules while an antibody has two antigen-binding sites.

Generally speaking, when two or more antibodies form an immune complexwith two or more antigens, the resulting immune complex can stronglybind to Fc receptors existing on cell surfaces due to avidity effectsthrough the Fc regions of the antibodies in the complex and can then betaken up into the cell with high efficiency. Thus, the above-mentionedanti-CCL2 antibody capable of forming an immune complex containing twoor more anti-CCL2 antibodies and two or more CCL2 molecules can lead toa rapid clearance of CCL2 from plasma in a living body, via the strongbinding to Fc receptors due to avidity effects.

Furthermore, an antibody with pH-dependent binding characteristics isthought to have superior properties in terms of antigen neutralizationand clearance relative to its counterpart that binds in a pH-independentmanner (Igawa et al., Nature Biotech. 28(11):1203-1207 (2010);Devanaboyina et al. mAbs 5(6):851-859 (2013); WO 2009/125825).Therefore, an antibody having both properties above, that is, anantibody which has pH-dependent binding characteristics and which formsan immune complex containing two or more antibodies with two or moreantigens, is expected to have even more superior properties for highlyaccelerated elimination of antigens from plasma (WO 2013/081143).

In one aspect, the invention provides polypeptides comprising variant Fcregions with enhanced FcgammaRIIb-binding activity comprising at leasttwo amino acid alterations comprising: (a) one amino acid alteration atposition 236, and (b) at least one amino acid alteration of at least oneposition selected from the group consisting of: 231, 232, 233, 234, 235,237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328,330, 331, 332, 334, and 396, according to EU numbering.

In one aspect, the invention provides polypeptides comprising a variantFc region with enhanced FcgammaRIIb-binding activity comprising an aminoacid alteration at position 236 according to EU numbering.

In one aspect, the invention provides polypeptides comprising a variantFc region with enhanced FcgammaRIIb-binding activity comprising at leasttwo amino acid alterations comprising: (a) one amino acid alteration atposition 236, and (b) at least one amino acid alteration of at least oneposition selected from the group consisting of: 231, 232, 235, 239, 268,295, 298, 326, 330, and 396, according to EU numbering. In a furtherembodiment, the variant Fc region comprises an amino acid alteration ofat least one position selected from the group consisting of: 231, 232,235, 239, 268, 295, 298, 326, 330, and 396, according to EU numbering.In a further embodiment, the variant Fc region comprises an amino acidalteration of at least one position selected from the group consistingof: 268, 295, 326, and 330, according to EU numbering.

In another aspect, the invention provides polypeptides comprisingvariant Fc regions with enhanced FcgammaRIIb-binding activity comprisingamino acid alterations of any one of the following (1)-(37): (1)positions 231, 236, 239, 268 and 330; (2) positions 231, 236, 239, 268,295 and 330; (3) positions 231, 236, 268 and 330; (4) positions 231,236, 268, 295 and 330; (5) positions 232, 236, 239, 268, 295 and 330;(6) positions 232, 236, 268, 295 and 330; (7) positions 232, 236, 268and 330; (8) positions 235, 236, 268, 295, 326 and 330; (9) positions235, 236, 268, 295 and 330; (10) positions 235, 236, 268 and 330; (11)positions 235, 236, 268, 330 and 396; (12) positions 235, 236, 268 and396; (13) positions 236, 239, 268, 295, 298 and 330; (14) positions 236,239, 268, 295, 326 and 330; (15) positions 236, 239, 268, 295 and 330;(16) positions 236, 239, 268, 298 and 330; (17) positions 236, 239, 268,326 and 330; (18) positions 236, 239, 268 and 330; (19) positions 236,239, 268, 330 and 396; (20) positions 236, 239, 268 and 396; (21)positions 236 and 268; (22) positions 236, 268 and 295; (23) positions236, 268, 295, 298 and 330; (24) positions 236, 268, 295, 326 and 330;(25) positions 236, 268, 295, 326, 330 and 396; (26) positions 236, 268,295 and 330; (27) positions 236, 268, 295, 330 and 396; (28) positions236, 268, 298 and 330; (29) positions 236, 268, 298 and 396; (30)positions 236, 268, 326 and 330; (31) positions 236, 268, 326, 330 and396; (32) positions 236, 268 and 330; (33) positions 236, 268, 330 and396; (34) positions 236, 268 and 396; (35) positions 236 and 295; (36)positions 236, 330 and 396; and (37) positions 236 and 396, according toEU numbering.

In a further embodiment, the variant Fc region with enhancedFcgammaRIIb-binding activity comprises at least one amino acid selectedfrom the group consisting of: (a) Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr at position 231;(b) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, Tyr at position 232; (c) Asp at position 233; (d)Trp, Tyr at position 234; (e) Trp at position 235; (f) Ala, Asp, Glu,His, Ile, Leu, Met, Asn, Gln, Ser, Thr, Val at position 236; (g) Asp,Tyr at position 237; (h) Glu, Ile, Met, Gln, Tyr at position 238; (i)Ile, Leu, Asn, Pro, Val at position 239; (j) Ile at position 264; (k)Phe at position 266; (1) Ala, His, Leu at position 267; (m) Asp, Glu atposition 268; (n) Asp, Glu, Gly at position 271; (o) Leu at position295; (p) Leu at position 298; (q) Glu, Phe, Ile, Leu at position 325;(r) Thr at position 326; (s) Ile, Asn at position 327; (t) Thr atposition 328; (u) Lys, Arg at position 330; (v) Glu at position 331; (w)Asp at position 332; (x) Asp, Ile, Met, Val, Tyr at position 334; and(y) Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, Tyr at position 396; according to EU numbering.

In a further embodiment, the variant Fc region with enhancedFcgammaRIIb-binding activity comprises at least one amino acidalteration (e.g., substitution) selected from the group consisting of:(a) Gly, Thr at position 231; (b) Asp at position 232; (c) Trp atposition 235; (d) Asn, Thr at position 236; (e) Val at position 239; (f)Asp, Glu at position 268; (g) Leu at position 295; (h) Leu at position298; (i) Thr at position 326; (j) Lys, Arg at position 330; and (k) Lys,Met at position 396; according to EU numbering. In a further embodiment,the variant Fc region with enhanced FcgammaRIIb-binding activitycomprises amino acid alterations (e.g., substitutions) of: Asn atposition 236, Glu at position 268, Lys at position 330, and Met atposition 396; according to EU numbering. In a further embodiment, thevariant Fc region with enhanced FcgammaRIIb-binding activity comprisesamino acid alterations (e.g., substitutions) of: Asn at position 236,Asp at position 268, and Lys at position 330; according to EU numbering.In a further embodiment, the variant Fc region with enhancedFcgammaRIIb-binding activity comprises amino acid alterations (e.g.,substitutions) of Asn at position 236, Asp at position 268, Leu atposition 295, and Lys at position 330; according to EU numbering. In afurther embodiment, the variant Fc region with enhancedFcgammaRIIb-binding activity comprises amino acid alterations (e.g.,substitutions) of Thr at position 236, Asp at position 268, and Lys atposition 330; according to EU numbering. In a further embodiment, thevariant Fc region with enhanced FcgammaRIIb-binding activity comprisesamino acid alterations (e.g., substitutions) of: Asn at position 236,Asp at position 268, Leu at position 295, Thr at position 326, and Lysat position 330; according to EU numbering. In a further embodiment, thevariant Fc region with enhanced FcgammaRIIb-binding activity comprisesamino acid alterations (e.g., substitutions) of Trp at position 235, Asnat position 236, Asp at position 268, Leu at position 295, Thr atposition 326, and Lys at position 330; according to EU numbering.

In another aspect, the invention provides isolated polypeptidescomprising variant Fc regions with increased isoelectric point (pI). Incertain embodiments, a variant Fc region described herein comprises atleast two amino acid alterations in a parent Fc region. In certainembodiments, each of the amino acid alterations increases theisoelectric point (pI) of the variant Fc region compared with that ofthe parent Fc region. They are based on the findings that antigenelimination from plasma can be promoted with an antibody whose pI hasbeen increased by modification of at least two amino acid residues, forexample when the antibody is administered in vivo.

In the present invention, pI may be either a theoretical or anexperimentally determined pI. The value of pI can be determined, forexample, by isoelectric focusing known to those skilled in the art. Thevalue of a theoretical pI can be calculated, for example, using gene andamino acid sequence analysis software (Genetyx, etc.).

In one embodiment, the pI value may be increased, for example, at leastby 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more, at least by 0.6,0.7, 0.8, 0.9, or more, at least by 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, ormore, or at least by 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,3.0 or more, as compared to before modification.

In certain embodiments, the amino acid for increased pI can be exposedon the surface of the variant Fc region. In the present invention, anamino acid that can be exposed on the surface generally refers to anamino acid residue located on the surface of a polypeptide constitutinga variant Fc region. An amino acid residue located on the surface of apolypeptide refers to an amino acid residue whose side chain can be incontact with solvent molecules (which in general are mostly watermolecules). However, the side chain does not necessarily have to bewholly in contact with solvent molecules, and when even a portion of theside chain is in contact with the solvent molecules, the amino acid isdefined as an “amino acid residue located on the surface”. The aminoacid residues located on the surface of a polypeptide also include aminoacid residues located close to the surface and thereby can have anelectric charge influence from another amino acid residue whose sidechain, even partly, is in contact with the solvent molecules. Thoseskilled in the art can prepare a homology model of a polypeptide forexample, using commercially available softwares. Alternatively, it ispossible to use methods known to those skilled in the art, such as X-raycrystallography. The amino acid residues that can be exposed on thesurface are determined, for example, using coordinates from athree-dimensional model using a computer program such as InsightIIprogram (Accelrys). Surface-exposable sites may be determined usingalgorithms known in the technical field (for example, Lee and Richards(J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558(1983)). Surface-exposable sites can be determined using softwaresuitable for protein modeling and three-dimensional structureinformation. Software available for such purposes includes, for example,the SYBYL Biopolymer Module software (Tripos Associates). When analgorithm requires a user input size parameter, the “size” of a probewhich is used in the calculation may be set to about 1.4 Angstrom orless in radius. Furthermore, methods for determining surface-exposableregions using software for personal computers have been described byPacios (Comput. Chem. 18(4):377-386 (1994); J. Mol. Model. 1:46-53(1995)). Based on such information as described above, appropriate aminoacid residues located on the surface of a polypeptide that constitutes avariant Fc region can be selected.

In certain embodiments, a polypeptide comprises both the variant Fcregion and an antigen-binding domain. In further embodiments, theantigen is a soluble antigen. In one embodiment, the antigen is presentin biological fluids (for example, plasma, interstitial fluid, lymphaticfluid, ascitic fluid, and pleural fluid) of subjects. The antigen mayalso be a membrane antigen.

In further embodiments, antigen-binding activity of the antigen-bindingdomain changes according to ion concentration conditions. In oneembodiment, ion concentration is not particularly limited and refers tohydrogen ion concentration (pH) or metal ion concentration. Herein,metal ions refer to ions of group I elements except hydrogen, such asalkaline metals and the copper group elements, group II elements such asalkaline earth metals and zinc group elements, group III elements exceptboron, group IV elements except carbon and silicon, group VIII elementssuch as iron group and platinum group elements, elements belonging tosubgroup A of groups V, VI, and VII, and metal elements such asantimony, bismuth, and polonium. In the present invention, metal ionsinclude, for example, calcium ion, as described in WO 2012/073992 and WO2013/125667. In one embodiment, “ion concentration condition” may be acondition that focuses on differences in the biological behavior of anantigen-binding domain between a low ion concentration and a high ionconcentration. Furthermore, “antigen-binding activity of anantigen-binding domain changes according to ion concentrationconditions” means that the antigen-binding activity of anantigen-binding domain changes between a low ion concentration and ahigh ion concentration (such an antigen-binding domain is referred toherein as “ion concentration-dependent antigen-binding domain”). Theantigen-binding activity of an antigen-binding domain under a high ionconcentration condition may be higher (stronger) or lower (weaker) thanthat under a low ion concentration condition. In one embodiment, ionconcentration-dependent antigen-binding domains (such as pH-dependentantigen-binding domains or calcium ion concentration-dependentantigen-binding domains) can be obtained by known methods, for example,described in WO 2009/125825, WO 2012/073992, and WO 2013/046722.

In the present invention, the antigen-binding activity of anantigen-binding domain under a high calcium ion concentration conditionmay be higher than under a low calcium ion concentration condition. Thehigh calcium ion concentration is not particularly limited to but may bea concentration selected between 100 micro M and 10 mM, between 200micro M and 5 mM, between 400 micro M and 3 mM, between 200 micro M and2 mM, between 400 micro M and 1 mM, or between 500 micro M and 2.5 mM,which is preferable to be close to the plasma (blood) concentration ofcalcium ion in vivo. Meanwhile, the low calcium ion concentration is notparticularly limited to but may be a concentration selected between 0.1micro M and 30 micro M, between 0.2 micro M and 20 micro M, between 0.5micro M and 10 micro M, between 1 micro M and 5 micro M, or between 2micro M and 4 micro M, which is preferable to be close to theconcentration of calcium ion in early endosomes in vivo.

In one embodiment, the ratio between the antigen-binding activitiesunder a low calcium ion concentration condition and a high calcium ionconcentration condition is not limited but the ratio of the dissociationconstant (KD) under a low calcium ion concentration condition to the KDunder a high calcium ion concentration condition, i.e., KD (low calciumion concentration condition)/KD (high calcium ion concentrationcondition), is 2 or more, 10 or more, or 40 or more. The upper limit ofthe ratio may be 400, 1000, or 10000, as long as such an antigen-bindingdomain can be produced by techniques known to those skilled in the art.Alternatively, for example, the dissociation rate constant (kd) can beused instead of the KD. In this case, the ratio of the kd under a lowcalcium ion concentration condition to the kd under a high calcium ionconcentration condition, i.e., kd (low calcium ion concentrationcondition)/kd (high calcium ion concentration condition), is 2 or more,5 or more, 10 or more, or 30 or more. The upper limit of the ratio maybe 50, 100, or 200, as long as the antigen-binding domain can beproduced based on the common technical knowledge of those skilled in theart.

In the present invention, the antigen-binding activity of anantigen-binding domain under a low hydrogen ion concentration (neutralpH) may be higher than under a high hydrogen ion concentration (acidicpH). The acidic pH may be, for example, a pH selected from pH4.0 topH6.5, selected from pH4.5 to pH6.5, selected from pH5.0 to pH6.5, orselected from pH5.5 to pH6.5, which is preferable to be close to the invivo pH in early endosomes. The acidic pH may also be, for example,pH5.8 or pH6.0. In particular embodiments, the acidic pH is pH5.8.Meanwhile, the neutral pH may be, for example, a pH selected from pH6.7to pH10.0, selected from pH6.7 to pH9.5, selected from pH7.0 to pH9.0,or selected from pH7.0 to pH8.0, which is preferable to be close to thein vivo pH in plasma (blood). The neutral pH may also be, for example,pH7.4 or pH7.0. In particular embodiments, the neutral pH is pH7.4.

In one embodiment, the ratio between the antigen-binding activitiesunder an acidic pH condition and a neutral pH condition is not limitedbut the ratio of the dissociation constant (KD) under an acidic pHcondition to the KD under a neutral pH condition, i.e., KD (acidic pHcondition)/KD (neutral pH condition), is 2 or more, 10 or more, or 40 ormore. The upper limit of the ratio may be 400, 1000, or 10000, as longas such an antigen-binding domain can be produced by techniques known tothose skilled in the art. Alternatively, for example, the dissociationrate constant (kd) can be used instead of the KD. In this case, theratio of the kd under an acidic pH condition to the kd under a neutralpH condition, i.e., kd (acidic pH condition)/kd (neutral pH condition)is 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit ofthe ratio may be 50, 100, or 200, as long as the antigen-binding domaincan be produced based on the common technical knowledge of those skilledin the art.

In one embodiment, for example, at least one amino acid residue issubstituted with an amino acid residue with a side-chain pKa of 4.0-8.0,and/or at least one amino acid with a side-chain pKa of 4.0-8.0 isinserted in the antigen-binding domain, as described in WO 2009/125825.The amino acid may be substituted and/or inserted at any site as long asthe antigen-binding activity of the antigen-binding domain becomesweaker under an acidic pH condition than under a neutral pH condition ascompared to before the substitution or insertion. When theantigen-binding domain has a variable region or CDR, the site may bewithin the variable region or CDR. The number of amino acids that aresubstituted or inserted can be appropriately determined by those skilledin the art; and the number may be one or more. Amino acids with aside-chain pKa of 4.0-8.0 can be used to change the antigen-bindingactivity of the antigen-binding domain according to the hydrogen ionconcentration condition. Such amino acids include, for example, naturalamino acids such as His (H) and Glu (E), and unnatural amino acids suchas histidine analogs (US2009/0035836), m-N02-Tyr (pKa 7.45), 3,5-Br2-Tyr(pKa 7.21), and 3,5-12-Tyr (pKa7.38) (Heyl et al., Bioorg. Med. Chem.11(17):3761-3768 (2003)). Amino acids with a side-chain pKa of 6.0-7.0can also be used, which include, e.g., His (H).

In another embodiment, preferable antigen-binding domains for thevariant Fc region with increased pI are described and can be obtained bymethods described in WO2016/125495 and WO2017/046994.

In certain embodiments, the variant Fc region with increased pIcomprises at least two amino acid alterations of at least two positionsselected from the group consisting of: 285, 311, 312, 315, 318, 333,335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413,420, 422, and 431, according to EU numbering.

In further embodiments, the variant Fc region with increased pIcomprises at least two amino acid alterations of at least two positionsselected from the group consisting of: 311, 341, 343, 384, 399, 400,401, 402, and 413, according to EU numbering.

In another aspect, the invention provides polypeptides comprisingvariant Fc regions with increased pI comprising amino acid alterationsof any one of the following (1)-(10): (1) positions 311 and 341; (2)positions 311 and 343; (3) positions 311, 343 and 413; (4) positions311, 384 and 413; (5) positions 311 and 399; (6) positions 311 and 401;(7) positions 311 and 413; (8) positions 400 and 413; (9) positions 401and 413; and (10) positions 402 and 413; according to EU numbering.

In one aspect, the invention provides polypeptides comprising variant Fcregions with enhanced FcgammaRIIb-binding activity and increased pIcomprising at least three amino acid alterations comprising: (a) atleast one amino acid alteration of at least one position selected fromthe group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239,264, 266, 267,268, 271, 295, 298, 325, 326, 327, 328, 330, 331, 332,334, and 396, according to EU numbering, and (b) at least two amino acidalterations of at least two positions selected from the group consistingof: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385,388, 390, 399, 400, 401, 402, 413, 420, 422, and 431, according to EUnumbering.

In one aspect, the invention provides polypeptides comprising variant Fcregions with enhanced FcgammaRIIb-binding activity and increased pI, andthat comprise at least three amino acid alterations comprising: (a) atleast one amino acid alteration of at least one position selected fromthe group consisting of: 231, 232, 235, 236, 239, 268, 295, 298, 326,330, and 396, according to EU numbering, and (b) at least two amino acidalterations of at least two positions selected from the group consistingof: 311, 341, 343, 384, 399, 400, 401, 402, and 413, according to EUnumbering.

In another aspect, the invention provides polypeptides comprisingvariant Fc regions with enhanced FcgammaRIIb-binding activity andincreased pI comprising amino acid alterations of any one of thefollowing (1)-(9): (1) positions 235, 236, 268, 295, 311, 326, 330 and343; (2) positions 236, 268, 295, 311, 326, 330 and 343; (3) positions236, 268, 295, 311, 330 and 413; (4) positions 236, 268, 311, 330, 396and 399; (5) positions 236, 268, 311, 330 and 343; (6) positions 236,268, 311, 330, 343 and 413; (7) positions 236, 268, 311, 330, 384 and413; (8) positions 236, 268, 311, 330 and 413; and (9) positions 236,268, 330, 396, 400 and 413; according to EU numbering.

In one aspect, the invention provides polypeptides comprising variant Fcregions with enhanced FcgammaRIIb-binding activity and increased pIcomprising at least three amino acid alterations comprising: (a) atleast one amino acid alteration of at least one position selected fromthe group consisting of: 234, 238, 250, 264, 267, 307, and 330, and (b)at least two amino acid alterations of at least two positions selectedfrom the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337,341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422,and 431, according to EU numbering. In further embodiments, thepolypeptides comprise at least two amino acid alterations of at leasttwo positions selected from the group consisting of: 311, 341, 343, 384,399, 400, 401, 402, and 413, according to EU numbering.

In another aspect, the invention provides polypeptides comprisingvariant Fc regions with enhanced FcgammaRIIb-binding activity andincreased pI comprising amino acid alterations of any one of thefollowing (1)-(16): (1) positions 234, 238, 250, 264, 307, 311, 330 and343; (2) positions 234, 238, 250, 264, 307, 311, 330 and 413; (3)positions 234, 238, 250, 264, 267, 307, 311, 330 and 343; (4) positions234, 238, 250, 264, 267, 307, 311, 330 and 413; (5) positions 234, 238,250, 267, 307, 311, 330 and 343; (6) positions 234, 238, 250, 267, 307,311, 330 and 413; (7) positions 234, 238, 250, 307, 311, 330 and 343;(8) positions 234, 238, 250, 307, 311, 330 and 413; (9) positions 238,250, 264, 267, 307, 311, 330 and 343; (10) positions 238, 250, 264, 267,307, 311, 330 and 413; (11) positions 238, 250, 264, 307, 311, 330 and343; (12) positions 238, 250, 264, 307, 311, 330 and 413; (13) positions238, 250, 267, 307, 311, 330 and 343; (14) positions 238, 250, 267, 307,311, 330 and 413; (15) positions 238, 250, 307, 311, 330 and 343; and(16) positions 238, 250, 307, 311, 330 and 413; according to EUnumbering.

In addition, amino acid alterations performed for other purpose(s) canbe combined in a variant Fc region described herein. For example, aminoacid substitutions that improve FcRn-binding activity (Hinton et al., J.Immunol. 176(1):346-356 (2006); Dall'Acqua et al., J. Biol. Chem.281(33):23514-23524 (2006); Petkova et al., Intl. Immunol.18(12):1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2):157-159(2010); WO 2006/019447; WO 2006/053301; and WO 2009/086320), and aminoacid substitutions for improving antibody heterogeneity or stability (WO2009/041613) may be added. Alternatively, polypeptides with the propertyof promoting antigen clearance, which are described in WO 2011/122011,WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides with theproperty of specific binding to a target tissue, which are described inWO 2013/180200, polypeptides with the property for repeated binding to aplurality of antigen molecules, which are described in WO 2009/125825,WO 2012/073992 or WO 2013/047752, can be combined with a variant Fcregion described herein. Alternatively, with the objective of conferringbinding ability to other antigens, the amino acid alterations disclosedin EP1752471 and EP1772465 may be combined in CH3 of a variant Fc regiondescribed herein. Alternatively, with the objective of increasing plasmaretention, amino acid alterations that decrease the pI of the constantregion (WO 2012/016227) may be combined in a variant Fc region describedherein. Alternatively, with the objective of promoting uptake intocells, amino acid alterations that increase the pI of the constantregion (WO 2014/145159) may be combined in a variant Fc region describedherein. Alternatively, with the objective of promoting elimination of atarget molecule from plasma, amino acid alterations that increase the pIof the constant region (WO2016/125495) may be combined in a variant Fcregion described herein. In one embodiment, such alteration may include,for example, substitution at al least one position selected from thegroup consisting of 311, 343, 384, 399, 400, and 413 according to EUnumbering. In a further embodiment, such substitution may be areplacement of an amino acid with Lys or Arg at each position.

Amino acid alterations of enhancing human FcRn-binding activity underacidic pH can also be combined in a variant Fc region described herein.Specifically, such alterations may include, for example, substitution ofLeu for Met at position 428 and substitution of Ser for Asn at position434, according to EU numbering (Zalevsky et al., Nat. Biotechnol.28:157-159 (2010)); substitution of Ala for Asn at position 434 (Deng etal., Metab. Dispos. 38(4):600-605 (2010)); substitution of Tyr for Metat position 252, substitution of Thr for Ser at position 254 andsubstitution of Glu for Thr at position 256 (Dall'Acqua et al., J. Biol.Chem. 281:23514-23524 (2006)); substitution of Gln for Thr at position250 and substitution of Leu for Met at position 428 (Hinton et al., J.Immunol. 176(1):346-356 (2006)); substitution of His for Asn at position434 (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011), andalterations described in WO 2010/106180, WO 2010/045193, WO 2009/058492,WO 2008/022152, WO 2006/050166, WO 2006/053301, WO 2006/031370, WO2005/123780, WO 2005/047327, WO 2005/037867, WO 2004/035752, or WO2002/060919. Such alterations may include, for example, at least onealteration selected from the group consisting of substitution of Leu forMet at position 428, substitution of Ala for Asn at position 434 andsubstitution of Thr for Tyr at position 436. Those alterations mayfurther include substitution of Arg for Gln at position 438 and/orsubstitution of Glu for Ser at position 440 (WO2016/125495).

Exemplary Bispecific-Anti-CCL2 Antibodies

-   One embodiment of the invention is a bispecific antibody comprising    a first antigen-binding site that (specifically) binds to a first    epitope on human CCL2 and a second different antigen-binding site    that (specifically) binds a second different epitope on human CCL2,    wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 49, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 50, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 51;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 52; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 53, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 54; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising a (d) CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 2, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 3;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 5, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 6; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG1 isotype.-   In one embodiment the in vivo clearance rate for human CCL2    (ml/day/kg) after administration of the bispecific antibody    comprising a constant heavy chain domain of human wild type IgG1    isotype (or the Fc domain thereof) is at least two fold higher (in    one embodiment at least 5 fold higher, in one embodiment at least 10    fold higher, in one embodiment at least 20 fold higher) compared to    the in vivo clearance rate for human CCL2 (ml/day/kg) after    administration of a bispecific antibody comprising a Fe gamma    receptor silenced constant heavy chain domain of human IgG1 isotype    (or the Fc domain thereof) comprising the mutations L234A, L235A,    P329G, when a pre-formed immune complex consisting of 20 mg/kg of    each bispecific antibody and 0.1 mg/kg human CCL2 was administered    at a single dose of 10 ml/kg into FcRn transgenic mice.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    -   wherein    -   i) said first antigen-binding site binds to same epitope on CCL2        as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and        -   ii) said second antigen-binding site binds to same epitope            on CCL2 as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG1 isotype.

In one embodiment the in vivo clearance rate for human CCL2 (ml/day/kg)after administration of the bispecific antibody comprising a constantheavy chain domain of human wild type IgG1 isotype (or the Fc domainthereof). is at least 15 fold higher, in particular at least 20 foldhigher, compared to the in vivo clearance rate for human CCL2(ml/day/kg) after administration of a bispecific antibody comprising aFc gamma receptor silenced constant heavy chain domain of human IgG1isotype (or the Fc domain thereof) comprising the mutations L234A,L235A, P329G, when a pre-formed immune complex consisting of 20 mg/kg ofeach bispecific antibody and 0.1 mg/kg human CCL2 was administered at asingle dose of 10 ml/kg into FcRn transgenic mice.

-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I or            T, (b) a CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62;        -   and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2            comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ            ID NO: 77 wherein X is D or E, and (c) a CDR-H3 comprising            the amino acid sequences GVFGFFXH of SEQ ID NO:78 wherein X            is D or E; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (e) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I or            T, (b) a CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, (c) a CDR-H3 comprising            the amino acid sequence YDAHYGELDF of SEQ ID NO: 59, (d) a            FR-H1 comprising the amino acid sequence            QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO:63, (e) a FR-H2            comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID            NO:64, (f) a FR-H3 comprising the amino acid sequence            RVTITADESTSTAYMELSSLRSEDTAVY YCAR of SEQ ID NO:65, and (g) a            FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ            ID NO:66;        -   and        -   a VL domain comprising (h) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (i) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (j) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62, (k) a FR-L1 comprising the            amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID            NO:67, (1) a FR-L2 comprising the amino acid sequence            WYQQKPGQAPRLLIY of SEQ ID NO:68, (m) a FR-L3 comprising the            amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ            ID NO:69, and (n) a FR-L4 comprising the amino acid sequence            GQGTKVEIK of SEQ ID NO:70;        -   and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2            comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ            ID NO: 77 wherein X is D or E, (c) a CDR-H3 comprising the            amino acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D            or E, (d) a FR-H1 comprising the amino acid sequence            QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO:82, (e) a FR-H2            comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID            NO:83, (f) a FR-H3 comprising the amino acid sequence            RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO:84, and (g) a            FR-H4 comprising the amino acid sequence WGQGTTVTVSS of SEQ            ID NO:85;        -   and        -   a VL domain comprising (h) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (i) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, (j) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R, (k) a FR-L1 comprising the amino            acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO:86, (1) a            FR-L2 comprising the amino acid sequence WYQQKPGKAPKLLIH of            SEQ ID NO:87, (m) a FR-L3 comprising the amino acid sequence            GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO:88, and (n) a            FR-L4 comprising the amino acid sequence FGGGTKVEIK of SEQ            ID NO:89.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   J) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   K) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   L) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   M) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   N) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   O) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   P) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG1 isotype.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    -   wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94.-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    -   wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG1 isotype. One embodiment of the invention    is an (isolated) bispecific antibody comprising a first    antigen-binding site that (specifically) binds to a first epitope on    human CCL2 and a second antigen-binding site that (specifically)    binds a second different epitope on human CCL2, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, X² is            P, and X³ is H, and (c) a CDR-H3 comprising the amino acid            sequence YDAHYGELDF of SEQ ID NO: 59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D, and (c) a CDR-H3 comprising the amino acid            sequences GVFGFFXH of SEQ ID NO:78 wherein X is D;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F and            X² is R, (e) a CDR-L2 comprising the amino acid sequence            GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the            amino acid sequence QQFXSAPYT of SEQ ID NO: 81 wherein X is            W;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, X² is            P, and X³ is H, and (c) a CDR-H3 comprising the amino acid            sequence YDAHYGELDF of SEQ ID NO: 59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D, and (c) a CDR-H3 comprising the amino acid            sequences GVFGFFXH of SEQ ID NO:78 wherein X is E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F and            X² is R, (e) a CDR-L2 comprising the amino acid sequence            GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the            amino acid sequence QQFXSAPYT of SEQ ID NO: 81 wherein X is            W;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   J) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   K) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   L) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   M) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   N) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   O) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   P) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG1 isotype.-   In one embodiment the bispecific antibody described herein    -   i) blocks binding of CCL2 to its receptor CCR2 in vitro        (reporter assay, IC₅₀=0.5 nM); and/or    -   ii) inhibits CCL2-mediated chemotaxis of myeloid cells in vitro        (IC₅₀=1.5 nM); and/or    -   iii) is cross-reactive to cynomolgus and human CCL2.-   In one embodiment the bispecific antibody described herein is not    cross-reactive to other human CCL homologs in particular it shows    100 time less binding to other CCL homologs (selected from the group    of CCL8, CCL7, and CCL13) compared to the binding to CCL2-   In one embodiment the bispecific antibody described herein binds to    the first and second epitope on human CCL2 in ion-dependent manner.-   In one embodiment the bispecific antibody described herein binds to    human CCL2 in pH dependent manner and wherein the first antigen    binding site and the second antigen binding site both bind to CCL2    with a higher affinity at neutral pH than at acidic pH.

In one embodiment the bispecific antibody described herein binds tohuman CCL2 with a 10 times higher affinity at pH 7.4, than at pH 5.8

In one embodiment the bispecific antibody described herein comprises twoIgG1 heavy chain constant domains (or the Fc domain thereof) comprising(independently or in addition to the above described mutations) thefollowing mutations (EU numbering)

-   -   i) S354C and T366W in one of the heavy chain constant domains    -   ii) Y349C, T366S, L368A, Y407V in the other of the heavy chain        constant domains

-   In one embodiment the bispecific antibody comprises a Fc domain of    human IgG1 isotype.

-   In one embodiment the bispecific antibody comprises constant heavy    chain domain of human IgG1 isotype. In one embodiment the bispecific    antibody described herein comprises a human IgG1 heavy chain    constant domain (or the Fc domain thereof) comprising one or more of    the following mutations (Kabat EU numbering)    -   i) Q311R and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L234Y, L235W, G236N, P238D, T250V, V264I, H268D, Q295L,        T307P, K326T and/or A330K (suitable for increasing affinity to        human FcgRIIb and decreasing affinity to other human FcgR);        and/or    -   iii) M428L, N434A and/or Y436T (suitable for increasing affinity        to FcRn for longer plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R, and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and/or    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iii) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering) Q311R and    P343R (suitable for increasing pI for enhancing uptake of antigen).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L234Y, P238D, T250V, V264I, T307P and/or A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and/or    -   iii) M428L, N434A and/or Y436T (suitable for increasing affinity        to FcRn for longer plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and iii) M428L, N434A and Y436T (suitable for        increasing affinity to FcRn for longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one embodiment the bispecific antibody described herein comprises    a human IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising one or more of the following mutations (Kabat EU    numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A and (suitable for increasing affinity to FcRn for        longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   wherein said bispecific antibody is a full length antibody of        human IgG isotype (preferably of human IgG1 isotype) with    -   a) a first light chain and a first heavy chain of a first        antibody comprising said first antigen binding site under the        respective i); and    -   b) a second light chain and a second heavy chain of a second        antibody comprising said second antigen binding site under the        respective ii), and wherein the variable domains VL and VH in        the second light chain and second heavy chain of the second        antibody are replaced by each other; and    -   wherein in the constant domain CL of the first light chain        under a) the amino acid at position 124 is substituted by        lysine (K) (numbering according to Kabat) and the amino acid at        position 123 is substituted by lysine (K) (numbering according        to Kabat),    -   and wherein in the constant domain CH1 of the first heavy chain        under a) the amino acid at position 147 is substituted by        glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index).

-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   wherein said bispecific antibody is a (full length) antibody        with    -   a) a first kappa or lambda light chain of and a first heavy        chain of IgG1 isotype comprising said first antigen binding site        under i); and    -   b) a second kappa or lambda light chain and a second IgG1 heavy        chain IgG1 isotype comprising said second antigen binding site        under ii), and wherein the variable domains VL and VH in the        second light chain and second heavy chain of the second antibody        are replaced by each other; and    -   wherein in the constant domain CL of the first light chain        under a) the amino acid at position 124 is substituted by        lysine (K) (numbering according to Kabat) and the amino acid at        position 123 is substituted by lysine (K) (numbering according        to Kabat),    -   and wherein in the constant domain CH1 of the first heavy chain        under a) the amino acid at position 147 is substituted by        glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index).

In one embodiment such bispecific antibody comprises the followingmutations (Kabat EU numbering)

-   -   i) S354C and T366W in one of the heavy chain constant domains    -   ii) Y349C, T366S, L368A, Y407V in the other of the heavy chain        constant domains

-   In one embodiment such bispecific antibody comprises in addition the    following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one alternative embodiment such bispecific antibody comprises in    addition the following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iii) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one alternative embodiment such bispecific antibody comprises in    addition the following mutations (Kabat EU numbering):    -   Q311R and P343R (suitable for increasing pI for enhancing uptake        of antigen).

-   In one alternative embodiment such bispecific antibody comprises in    addition the following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) M428L, N434A and Y436T (suitable for increasing affinity to        FcRn for longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding.

-   In one alternative embodiment such bispecific antibody comprises in    addition the following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A and (suitable for increasing affinity to FcRn for        longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 112, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 113, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 114, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 115.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 112, a polypeptide comprising the amino acidsequence of SEQ ID NO: 113, a polypeptide comprising the amino acidsequence of SEQ ID NO: 114 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 115.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 116, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 117, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 118, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 119.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 116, a polypeptide comprising the amino acidsequence of SEQ ID NO: 117, a polypeptide comprising the amino acidsequence of SEQ ID NO: 118 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 119.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 120, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 121, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 122, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 123.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 120, a polypeptide comprising the amino acidsequence of SEQ ID NO: 121, a polypeptide comprising the amino acidsequence of SEQ ID NO: 122 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 123.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 120, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 121, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 122, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 123.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 120, a polypeptide comprising the amino acidsequence of SEQ ID NO: 121, a polypeptide comprising the amino acidsequence of SEQ ID NO: 122 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 123.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 155, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 156, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 157, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 158.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 155, a polypeptide comprising the amino acidsequence of SEQ ID NO: 156, a polypeptide comprising the amino acidsequence of SEQ ID NO: 157 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 158.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 159, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 160, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 161, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 162.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 159, a polypeptide comprising the amino acidsequence of SEQ ID NO: 160, a polypeptide comprising the amino acidsequence of SEQ ID NO: 161 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 162.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 163, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 164, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 165, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 166.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 163, a polypeptide comprising the amino acidsequence of SEQ ID NO: 164, a polypeptide comprising the amino acidsequence of SEQ ID NO: 165 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 166.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 167, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 168, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 169, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 170.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 167, a polypeptide comprising the amino acidsequence of SEQ ID NO: 168, a polypeptide comprising the amino acidsequence of SEQ ID NO: 169 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 170.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 171, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 172, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 173, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 174.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 171, a polypeptide comprising the amino acidsequence of SEQ ID NO: 172, a polypeptide comprising the amino acidsequence of SEQ ID NO: 173 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 174.

-   One embodiment of the invention is an (isolated) bispecific antibody    comprising a first antigen-binding site that (specifically) binds to    a first epitope on human CCL2 and a second antigen-binding site that    (specifically) binds a second different epitope on human CCL2,    wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   wherein said bispecific antibody is a (full length) antibody of        human IgG1 isotype with    -   a) a first kappa or lambda light chain of and a first heavy        chain of IgG1 isotype comprising said first antigen binding site        under i); and    -   b) a second kappa or lambda light chain and a second IgG1 heavy        chain IgG1 isotype comprising said second antigen binding site        under ii), and wherein the variable domains VL and VH in the        second light chain and second heavy chain of the second antibody        are replaced by each other; and    -   wherein in the constant domain CL of the first light chain        under a) the amino acid at position 124 is substituted by        lysine (K) (numbering according to Kabat) and the amino acid at        position 123 is substituted by lysine (K) (numbering according        to Kabat),    -   and wherein in the constant domain CH1 of the first heavy chain        under a) the amino acid at position 147 is substituted by        glutamic acid (E) (numbering according to Kabat EU index) and        the amino acid at position 213 is substituted by glutamic        acid (E) (numbering according to Kabat EU index).

In one embodiment such bispecific antibody comprises the followingmutations (Kabat EU numbering)

-   -   i) S354C and T366W in one of the heavy chain constant domains    -   ii) Y349C, T366S, L368A, Y407V in the other of the heavy chain        constant domains

-   In one embodiment such bispecific antibody comprises in addition the    following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iii) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one embodiment the bispecific antibody described herein comprises    a IgG1 heavy chain constant domain (or the Fc domain thereof)    comprising the following mutations (Kabat EU numbering) Q311R and    P343R (suitable for increasing pI for enhancing uptake of antigen).

-   In one alternative embodiment such bispecific antibody comprises in    addition the following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) M428L, N434A and Y436T (suitable for increasing affinity to        FcRn for longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

-   In one alternative embodiment such bispecific antibody comprises in    addition the following mutations (Kabat EU numbering):    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307P and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A and (suitable for increasing affinity to FcRn for        longer plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 124, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 125, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 126, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 127.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 124, a polypeptide comprising the amino acidsequence of SEQ ID NO: 125, a polypeptide comprising the amino acidsequence of SEQ ID NO: 126 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 127.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 128, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 129, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 130, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 131.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 128, a polypeptide comprising the amino acidsequence of SEQ ID NO: 129, a polypeptide comprising the amino acidsequence of SEQ ID NO: 130 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 131.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 wherein thebispecific antibody comprises a polypeptide comprising an amino acidsequence that is at least 98%, or 99% identical to the sequence of SEQID NO: 132, a polypeptide comprising an amino acid sequence that is atleast 98%, or 99% identical to the sequence of SEQ ID NO: 133, apolypeptide comprising an amino acid sequence that is at least 98%, or99% identical to the sequence of SEQ ID NO: 134, and a polypeptidecomprising an amino acid sequence that is at least 98%, or 99% identicalto the sequence of SEQ ID NO: 135.

A specific embodiment of the invention is an (isolated) bispecificantibody comprising a first antigen-binding site that (specifically)binds to a first epitope on human CCL2 and a second antigen-binding sitethat (specifically) binds a second epitope on human CCL2 whereinbispecific antibody comprises a polypeptide comprising the amino acidsequence of SEQ ID NO: 132, a polypeptide comprising the amino acidsequence of SEQ ID NO: 133, a polypeptide comprising the amino acidsequence of SEQ ID NO: 134 and a polypeptide comprising the amino acidsequence of SEQ ID NO: 135.

Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CCL2 antibody (either bispecificor monospecific) as described herein is provided. Such nucleic acid mayencode an amino acid sequence comprising one or all VL and/or an aminoacid sequence comprising one or all VH of the mono- or bispecificantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell, a HEK293cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, amethod of making an anti-CCL2 antibody is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of an anti-CCL2 cell, such nucleic acid maybe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli.) After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHOcells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

In another aspect, the invention is based, in part, on the finding thatthe modified monospecific antibodies as described herein show improvedpH dependent binding properties and re therefore especially useful forthe generation of the bispecific antibodies of the invention

Monospecific Anti-CCL2 Antibodies with pH Dependent Binding Properties

-   One embodiment of the invention is an (isolated) (monospecific)    antibody that (specifically) binds to a human CCL2, wherein the    antibody comprises    -   A) a VH domain comprising (a) a CDR-H1 comprising the amino acid        sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a        CDR-H2 comprising the amino acid sequence GX¹IX²IFX³TANYAQKFQG        of SEQ ID NO: 58 wherein X¹ is V, I, or H, X² is P or H, and X³        is H or G, and (c) a CDR-H3 comprising the amino acid sequence        YDAHYGELDF of SEQ ID NO: 59;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62;        -   or    -   B) a VH domain comprising (a) a CDR-H1 comprising the amino acid        sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the        amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77 wherein X        is D or E, and (c) a CDR-H3 comprising the amino acid sequences        GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (e) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   One embodiment of the invention is an (isolated) (monospecific)    antibody that (specifically) binds to a human CCL2, wherein the    antibody comprises    -   A) a VH domain comprising the amino acid sequence of SEQ ID        NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   B) a VH domain comprising the amino acid sequence of SEQ ID        NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   C) a VH domain comprising the amino acid sequence of SEQ ID        NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   D) a VH domain comprising the amino acid sequence of SEQ ID        NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   E) a VH domain comprising the amino acid sequence of SEQ ID        NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   F) a VH domain comprising the amino acid sequence of SEQ ID        NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   G) a VH domain comprising the amino acid sequence of SEQ ID        NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   H) a VH domain comprising the amino acid sequence of SEQ ID        NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;            Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the mono- or bispecific anti-CCL2antibodies provided herein is useful for detecting the presence of CCL2in a biological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as immune cell or Tcell infiltrates and or tumor cells.

In one embodiment, a mono- or bispecific anti-CCL2 antibody for use in amethod of diagnosis or detection is provided. In a further aspect, amethod of detecting the presence of CCL2 in a biological sample isprovided. In certain embodiments, the method comprises contacting thebiological sample with a mono- or bispecific anti-CCL2 antibody asdescribed herein under conditions permissive for binding of the mono- orbispecific anti-CCL2 antibody to CCL2, and detecting whether a complexis formed between the mono- or bispecific anti-CCL2 antibody and CCL2.Such method may be an in vitro or in vivo method. In one embodiment, amono- or bispecific anti-CCL2 antibody is used to select subjectseligible for therapy with a mono- or bispecific anti-CCL2 antibody, e.g.where CCL2 is a biomarker for selection of patients.

In certain embodiments, labeled mono- or bispecific anti-CCL2 antibodiesare provided. Labels include, but are not limited to, labels or moietiesthat are detected directly (such as fluorescent, chromophoric,electron-dense, chemiluminescent, and radioactive labels), as well asmoieties, such as enzymes or ligands, that are detected indirectly,e.g., through an enzymatic reaction or molecular interaction. Exemplarylabels include, but are not limited to, the radioisotopes ³²P, ¹⁴C,¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates orfluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, luceriferases, e.g., firefly luciferase and bacterialluciferase (U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkalinephosphatase, p-galactosidase, glucoamylase, lysozyme, saccharideoxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

E. Pharmaceutical Formulations

Pharmaceutical formulations of a mono- or bispecific anti-CCL2 antibodyas described herein are prepared by mixing such antibody having thedesired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences, 16th edition,Osol, A. (ed.) (1980)), in the form of lyophilized formulations oraqueous solutions. Pharmaceutically acceptable carriers are generallynontoxic to recipients at the dosages and concentrations employed, andinclude, but are not limited to: buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyl dimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride; benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone);amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rhuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methyl methacrylate) microcapsules, respectively, in colloidaldrug delivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

F. Therapeutic Methods and Compositions

Any of the mono- or bispecific anti-CCL2 antibodies provided herein maybe used in therapeutic methods.

In one aspect, a mono- or bispecific anti-CCL2 antibody for use as amedicament is provided. In further aspects, a mono- or bispecificanti-CCL2 antibody or use in treating cancer is provided. In certainembodiments, a mono- or bispecific anti-CCL2antibody for use in a methodof treatment is provided. In certain embodiments, the invention providesa mono- or bispecific anti-CCL2 antibody for use in a method of treatingan individual having cancer comprising administering to the individualan effective amount of the mono- or bispecific anti-CCL2 antibody.

In further embodiments, the invention provides a mono- or bispecificanti-CCL2 antibody inhibits immunosuppression in tumors and thus makestumor susceptible for immuno stimulatory agents like anti-PD1,anti-PDL-1 antagonists and the like.

Therefore one aspect of the is the combination of the mono- orbispecific anti-CCL2 antibodies described here with a cancerimmunotherapy like anti-PD1, anti-PDL-1 antagonists and the like.

The term “cancer” as used herein may be, for example, lung cancer, nonsmall cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colon cancer, breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, mesothelioma, hepatocellular cancer, biliarycancer, neoplasms of the central nervous system (CNS), spinal axistumors, brain stem glioma, glioblastoma multiforme, astrocytomas,schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cellcarcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, includingrefractory versions of any of the above cancers, or a combination of oneor more of the above cancers.

An “individual” according to any of the above embodiments is preferablya human. In a further aspect, the invention provides for the use of amono- or bispecific anti-CCL2 antibody in the manufacture or preparationof a medicament. In one embodiment, the medicament is for treatment ofcancer. In a further embodiment, the medicament is for use in a methodof treating cancer comprising administering to an individual havingcancer an effective amount of the medicament. In a further embodiment,the medicament is for inducing cell mediated lysis of cancer cells In afurther embodiment, the medicament is for use in a method of inducingcell mediated lysis of cancer cells in an individual suffering fromcancer comprising administering to the individual an amount effective ofthe medicament to induce apoptosis in a cancer cell/or to inhibit cancercell proliferation. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for treatingcancer. In one embodiment, the method comprises administering to anindividual having cancer an effective amount of mono- or bispecificanti-CCL2 antibody. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for inducing cellmediated lysis of cancer cells in an individual suffering from cancer.In one embodiment, the method comprises administering to the individualan effective amount of a mono- or bispecific anti-CCL2 antibody toinduce cell mediated lysis of cancer cells in the individual sufferingfrom cancer. In one embodiment, an “individual” is a human.

In another aspect of the invention, a mono- or bispecific anti-CCL2antibody for use in treating inflammatory diseases or autoimmunediseases is provided. In certain embodiments, the invention provides amono- or bispecific anti-CCL2 antibody for use in a method of treatingan individual having an inflammatory disease or autoimmune diseasecomprising administering to the individual an effective amount of themono- or bispecific anti-CCL2 antibody.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the mono- or bispecific anti-CCL2 antibodies providedherein, e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical formulation comprises any of the mono- orbispecific anti-CCL2 antibodies provided herein and a pharmaceuticallyacceptable carrier.

In some embodiments, the inflammatory diseases or autoimmune disease isan autoimmune disorder, inflammatory disorder, fibrotic disorder,granulocytic (neutrophilic or eosinophilic) disorder, monocyticdisorder, or lymphocytic disorder, or a disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia). In some embodiments, the disorder is a pulmonary disorder.In some embodiments the pulmonary disorder is associated withgranulocytic (eosinophilic and/or neutrophilic) pulmonary inflammation,infection-induced pulmonary conditions (including those associated withviral (e.g., influenza, parainfluenza, rhinovirus, humanmetapneumovirus, and respiratory syncytial virus), bacterial, or fungal(e.g., Aspergillus) triggers. In some embodiments, the disorder is anallergen-induced pulmonary condition, a toxic environmentalpollutant-induced pulmonary condition (e.g., asbestosis, silicosis, orberylliosis), a gastric aspiration-induced pulmonary condition, orassociated with immune dysregulation or an inflammatory condition withgenetic predisposition such as cystic fibrosis. In some embodiments, thedisorder is a physical trauma-induced pulmonary condition (e.g.,ventilator injury), emphysema, cigarette-induced emphysema, bronchitis,sarcoidosis, histiocytosis, lymphangiomyomatosis, acute lung injury,acute respiratory distress syndrome, chronic lung disease,bronchopulmonary dysplasia, pneumonia (e.g., community-acquiredpneumonia, nosocomial pneumonia, ventilator-associated pneumonia, viralpneumonia, bacterial pneumonia, and severe pneumonia), airwayexacerbations, and acute respiratory distress syndrome (ARDS)). In someembodiments, the inflammatory pulmonary disorder is COPD.

In some embodiments, the inflammatory pulmonary disorder is asthma. Insome embodiments, the asthma is persistent chronic severe asthma withacute events of worsening symptoms (exacerbations or flares) that can belife threatening. In some embodiments, the asthma is atopic (also knownas allergic) asthma, non-allergic asthma (e.g., often triggered byinfection with a respiratory virus (e.g., influenza, parainfluenza,rhinovirus, human metapneumovirus, and respiratory syncytial virus) orinhaled irritant (air pollutants, smog, diesel particles, volatilechemicals and gases indoors or outdoors, or even by cold dry air),

In some embodiments, the asthma is intermittent or exercise-induced,asthma due to acute or chronic primary or second-hand exposure to“smoke” (typically cigarettes, cigars, pipes), inhaling or “vaping”(tobacco, marijuana or other such substances), or asthma triggered byrecent ingestion of aspirin or related NSAIDS. In some embodiments, theasthma is mild, or corticosteroid naïve asthma, newly diagnosed anduntreated asthma, or not previously requiring chronic use of inhaledtopical or systemic steroids to control the symptoms (cough, wheeze,shortness of breath/breathlessness, or chest pain). IN some embodiments,the asthma is chronic, corticosteroid resistant asthma, corticosteroidrefractory asthma, asthma uncontrolled on corticosteroids or otherchronic asthma controller medications. In some embodiments, theautoimmune disorder, inflammatory disorder, fibrotic disorder,neutrophilic disorder, or eosinophilic disorder is pulmonary fibrosis.In some embodiments, the pulmonary fibrosis is idiopathic pulmonaryfibrosis (IPF). In some embodiments, the autoimmune disorder,inflammatory disorder, fibrotic disorder, granulocytic (neutrophilic oreosinophilic) disorder, monocytic disorder, or lymphocytic disorder isesophogitis, allergic rhinitis, non-allergic rhinitis, rhinosinusitiswith polyps, nasal polyposis, bronchitis, chronic pneumonia, allergicbronchopulmonary aspergillosis, airway inflammation, allergic rhinitis,bronchiectasis, and/or chronic bronchitis.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder, is arthritis. In someembodiments, the arthritis is rheumatoid arthritis. In some embodiments,the arthritis is osteoarthritis, rheumatoid arthritis, juvenilearthritis, juvenile rheumatoid arthritis, early arthritis, polyarticularrheumatoid arthritis, systemic-onset rheumatoid arthritis, enteropathicarthritis, reactive arthritis, psoriatic arthritis, and/or arthritis asa result of injury.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder is a gastrointestinalinflammatory condition. In some embodiments, the gastrointestinalinflammatory condition is IBD (inflammatory bowel disease), ulcerativecolitis (UC), Crohn's disease (CD), colitis (e.g., colitis caused byenvironmental insults (e.g., caused by or associated with a therapeuticregimen, such as chemotherapy, radiation therapy, etc.), infectiouscolitis, ischemic colitis, collagenous or lymphocytic colitis,necrotizing enterocolitis, colitis in conditions such as chronicgranulomatous disease or celiac disease, food allergies, gastritis,gastroenteritis, infectious gastritis or enterocolitis (e.g.,Helicobacter pylori-infected chronic active gastritis), and other formsof gastrointestinal inflammation caused by an infectious agent, orindeterminate colitis.

In some embodiments, the autoimmune disorder, inflammatory disorder,fibrotic disorder, granulocytic (neutrophilic or eosinophilic) disorder,monocytic disorder, or lymphocytic disorder, or disorder associated withincreased numbers or distribution of normal or aberrant tissue residentcells (such as mast cells, macrophages, or lymphocytes) or stromal cells(such as fibroblasts, myofibroblasts, smooth muscle cells, epithelia, orendothelia) is lupus or Systemic Lupus Erythematosus (SLE), or one ormore organ-specific manifestations of lupus (e.g., lupus nephritis (LN)affecting the kidney, or extra-renal lupus (ERL) affecting the bloodand/or lymphoid organs (lymph nodes, spleen, thymus, and associatedlymphatic vessels), and/or joints and/or other organs, but notnecessarily the kidney). In some embodiments, the autoimmune disorder,inflammatory disorder, or fibrotic disorder is related to sepsis and/ortrauma, HIV infection, or idiopathic (of unknown etiology) such asANCA-associated vaculitides (AAV), granulomatosis with polyangiitis(formerly known as Wegener's granulomatosis), Behcet's disease,cardiovascular disease, eosinophilic bronchitis, Reiter's Syndrome, SEASyndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),ankylosing spondylitis, dermatomyositis, scleroderma, e.g., systemicscleroderma also called systemic sclerosis, vasculitis (e.g., Giant CellArteritis (GCA), also called temporal arteritis, cranial arteritis orHorton disease), myositis, polymyositis, dermatomyositis, polyarteritisnodosa, arteritis, polymyalgia rheumatica, sarcoidosis, primary biliarysclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaquepsoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis,erythrodermic psoriasis, dermatitis, atopic dermatitis, pemphigus, e.g.,pemphigus vulgaris, atherosclerosis, lupus, Still's disease, myastheniagravis, celiac disease, multiple sclerosis (MS) of therelapsing-remitting (RRMS) or primary progressive (PPMS) or secondaryprogressive (SPMS) subtypes, Guillain-Barre disease, Type I diabetesmellitus (T1DM) or insulin-dependent (IDDM) or juvenile onset DM type,thyroiditis (e.g., Graves' disease), coeliac disease, Churg-Strausssyndrome, myalgia syndrome, hypereosinophilic syndrome, oedematousreactions including episodic angioedema, helminth infections,onchocercal dermatitis eosinophilic oesophagitis, eosinophilicenteritis, eosinophilic colitis, obstructive sleep apnea, endomyocardialfibrosis, Addison's disease, Raynaud's disease or phenomenon, autoimmunehepatitis, graft versus host disease (GVHD), or organ transplantrejection.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intra-arterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.5 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

II. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

In the following specific embodiments of the invention are listed:

-   1. A bispecific antibody comprising a first antigen-binding site    that (specifically) binds to a first epitope on human CCL2 and a    second different antigen-binding site that (specifically) binds a    second different epitope on human CCL2, wherein the bispecific    antibody comprises a Fc domain of human IgG isotype, preferably of    IgG1 isotype.-   2. The bispecific antibody according to embodiment 1, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 33, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 34, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 35;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 36; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 37, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 49, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 50, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 51;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 52; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 53, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 54; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 41, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 42, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 43; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 44; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 45, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 46;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 17, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 18, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 19;        -   and a VL domain comprising a (d) CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 20; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 21, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 22;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 9, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 10, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 11;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 12; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 13, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 2, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 3;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 4; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 5, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 6; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence of SEQ ID NO: 25, (b) a CDR-H2 comprising the            amino acid sequence of SEQ ID NO: 26, and (c) a CDR-H3            comprising the amino acid sequence of SEQ ID NO: 27;        -   and a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence of SEQ ID NO: 28; (e) a CDR-L2 comprising the            amino acid sequence of SEQ ID NO: 29, and (f) a CDR-L3            comprising the amino acid sequence of SEQ ID NO: 30.-   3. The bispecific antibody according to embodiment 2, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:23 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 17, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 19;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:24 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 20; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 22;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:15 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 9, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 11;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:16 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 12; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 14;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:23 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 17, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 19;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:24 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 20; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 22; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:31 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 25, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 27;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:32 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 28; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 30; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46;    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:55 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 49, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 51;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:56 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 52; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 54; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:15 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 9, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 11;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:16 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 12; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:23 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 17, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 19;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:24 wherein the VL domain comprises a (d) CDR-L1            comprising the amino acid sequence of SEQ ID NO: 20; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 22;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:15 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 9, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 11;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:16 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 12; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 14; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:31 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 25, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 27;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:32 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 28; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 30;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:7 wherein the VH domain comprises (a) a CDR-H1 comprising            the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2            comprising the amino acid sequence of SEQ ID NO: 2, and (c)            a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:8 wherein the VL domain comprises (d) a CDR-L1 comprising            the amino acid sequence of SEQ ID NO: 4; (e) a CDR-L2            comprising the amino acid sequence of SEQ ID NO: 5, and (f)            a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6;            and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:31 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 25, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 27;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:32 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 28; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 30.-   4. The bispecific antibody according to any one of claims 1 to 3,    wherein the bispecific antibody comprising a Fc domain of human IgG    isotype is a bispecific antibody comprising a constant heavy chain    domain of human IgG1 isotype.-   5. The bispecific antibody according to embodiment 4, wherein the in    vivo clearance rate for human CCL2 (ml/day/kg) after administration    of the bispecific antibody comprising a constant heavy chain domain    of human wild type IgG1 isotype is at least two fold higher (in one    embodiment at least 5 fold higher, in one embodiment at least 10    fold higher, in one embodiment at least 20 fold higher) compared to    the in vivo clearance rate for human CCL2 (ml/day/kg) after    administration of a bispecific antibody comprising a Fc gamma    receptor silenced constant heavy chain domain of human IgG1 isotype    comprising the mutations L234A, L235A, P329G (Kabat EU numbering),    when a pre-formed immune complex consisting of 20 mg/kg of each    bispecific antibody and 0.1 mg/kg human CCL2 was administered at a    single dose of 10 ml/kg into FcRn transgenic mice.-   6. An (isolated) bispecific antibody comprising a first    antigen-binding site that (specifically) binds to a first epitope on    human CCL2 and a second antigen-binding site that (specifically)    binds a second different epitope on human CCL2,    -   wherein the bispecific antibody comprises a Fc domain of human        IgG isotype and wherein    -   i) said first antigen-binding site binds to same epitope on CCL2        as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:39 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 33, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 35;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:40 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 36; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 38; and    -   ii) said second antigen-binding site binds to same epitope on        CCL2 as an antibody comprising        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:47 wherein the VH domain comprises (a) a CDR-H1            comprising the amino acid sequence of SEQ ID NO: 41, (b) a            CDR-H2 comprising the amino acid sequence of SEQ ID NO: 42,            and (c) a CDR-H3 comprising the amino acid sequence of SEQ            ID NO: 43;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:48 wherein the VL domain comprises (d) a CDR-L1            comprising the amino acid sequence of SEQ ID NO: 44; (e) a            CDR-L2 comprising the amino acid sequence of SEQ ID NO: 45,            and (f) a CDR-L3 comprising the amino acid sequence of SEQ            ID NO: 46.-   7. The bispecific antibody according to embodiment 6, wherein the in    vivo clearance rate for human CCL2 (ml/day/kg) after administration    of the bispecific antibody comprising a constant heavy chain domain    of human wild type IgG1 isotype is at least 15 fold higher, in    particular at least 20 fold higher, compared to the in vivo    clearance rate for human CCL2 (ml/day/kg) after administration of a    bispecific antibody comprising a Fc gamma receptor silenced constant    heavy chain domain of human IgG1 isotype comprising the mutations    L234A, L235A, P329G (Kabat EU numbering), when a pre-formed immune    complex consisting of 20 mg/kg of each bispecific antibody and 0.1    mg/kg human CCL2 was administered at a single dose of 10 ml/kg into    FcRn transgenic mice.-   8. An (isolated) bispecific antibody comprising a first    antigen-binding site that (specifically) binds to a first epitope on    human CCL2 and a second antigen-binding site that (specifically)    binds a second epitope on human CCL2, wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I or            T, (b) a CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62;        -   and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2            comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ            ID NO: 77 wherein X is D or E, and (c) a CDR-H3 comprising            the amino acid sequences GVFGFFXH of SEQ ID NO:78 wherein X            is D or E;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (e) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   9. An (isolated) bispecific antibody comprising a first    antigen-binding site that (specifically) binds to a first epitope on    human CCL2 and a second antigen-binding site that (specifically)    binds a second epitope on human CCL2, wherein    -   i) said first antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence SHYGXS of SEQ ID NO: 57 wherein X is I or            T, (b) a CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, (c) a CDR-H3 comprising            the amino acid sequence YDAHYGELDF of SEQ ID NO: 59, (d) a            FR-H1 comprising the amino acid sequence            QVQLVQSGAEVKKPGSSVKVSCKASGGTF of SEQ ID NO:63, (e) a FR-H2            comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID            NO:64, (f) a FR-H3 comprising the amino acid sequence            RVTITADESTSTAYMELSSLRSEDTAVY YCAR of SEQ ID NO:65, and (g) a            FR-H4 comprising the amino acid sequence WGQGTLVTVSS of SEQ            ID NO:66;        -   and        -   a VL domain comprising (h) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (i) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (j) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62, (k) a FR-L1 comprising the            amino acid sequence EIVLTQSPATLSLSPGERATLSC of SEQ ID            NO:67, (1) a FR-L2 comprising the amino acid sequence            WYQQKPGQAPRLLIY of SEQ ID NO:68, (m) a FR-L3 comprising the            amino acid sequence GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC of SEQ            ID NO:69, and (n) a FR-L4 comprising the amino acid sequence            GQGTKVEIK of SEQ ID NO:70;        -   and    -   ii) said second antigen-binding site comprises        -   a VH domain comprising (a) a CDR-H1 comprising the amino            acid sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2            comprising the amino acid sequence RIDPXNHNTKFDPKFQG of SEQ            ID NO: 77 wherein X is D or E, (c) a CDR-H3 comprising the            amino acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D            or E, (d) a FR-H1 comprising the amino acid sequence            QVQLVQSGAEVKKPGSSVKVSCKASGLTIS of SEQ ID NO:82, (e) a FR-H2            comprising the amino acid sequence WVRQAPGQGLEWMG of SEQ ID            NO:83, (f) a FR-H3 comprising the amino acid sequence            RVTITADTSTSTAYMELSSLRSEDTAVYYCAR of SEQ ID NO:84, and (g) a            FR-H4 comprising the amino acid sequence WGQGTTVTVSS of SEQ            ID NO:85;        -   and        -   a VL domain comprising (h) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (i) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, (j) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R, (k) a FR-L1 comprising the amino            acid sequence DIQMTQSPSSLSASVGDRVTITC of SEQ ID NO:86, (1) a            FR-L2 comprising the amino acid sequence WYQQKPGKAPKLLIH of            SEQ ID NO:87, (m) a FR-L3 comprising the amino acid sequence            GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC of SEQ ID NO:88, and (n) a            FR-L4 comprising the amino acid sequence FGGGTKVEIK of SEQ            ID NO:89.-   10. An (isolated) bispecific antibody comprising a first    antigen-binding site that (specifically) binds to a first epitope on    human CCL2 and a second antigen-binding site that (specifically)    binds a second epitope on human CCL2, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   J) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   K) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   L) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   M) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;    -   or    -   N) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   O) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   P) i) said first antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75; and        -   ii) said second antigen-binding site comprises        -   a VH domain comprising the amino acid sequence of SEQ ID            NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93.-   11. An (isolated) bispecific antibody comprising a first    antigen-binding site that (specifically) binds to a first epitope on    human CCL2 and a second antigen-binding site that (specifically)    binds a second epitope on human CCL2, wherein    -   A) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, X² is            P, and X³ is H, and (c) a CDR-H3 comprising the amino acid            sequence YDAHYGELDF of SEQ ID NO: 59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D, and (c) a CDR-H3 comprising the amino acid            sequences GVFGFFXH of SEQ ID NO:78 wherein X is D;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F and            X² is R, (e) a CDR-L2 comprising the amino acid sequence            GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the            amino acid sequence QQFXSAPYT of SEQ ID NO: 81 wherein X is            W;    -   or    -   B) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, X² is            P, and X³ is H, and (c) a CDR-H3 comprising the amino acid            sequence YDAHYGELDF of SEQ ID NO: 59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D, and (c) a CDR-H3 comprising the amino acid            sequences GVFGFFXH of SEQ ID NO:78 wherein X is E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F and            X² is R, (e) a CDR-L2 comprising the amino acid sequence            GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3 comprising the            amino acid sequence QQFXSAPYT of SEQ ID NO: 81 wherein X is            W;    -   or    -   C) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   D) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   E) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   F) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   G) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   H) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:73 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   I) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   J) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   K) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:72 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   L) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   M) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:90 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:94 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   N) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   O) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:74 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:91 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R;    -   or    -   P) i) said first antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:71 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a            CDR-H2 comprising the amino acid sequence            GX¹IX²IFX³TANYAQKFQG of SEQ ID NO: 58 wherein X¹ is V, I, or            H, X² is P or H, and X³ is H or G, and (c) a CDR-H3            comprising the amino acid sequence YDAHYGELDF of SEQ ID NO:            59;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:75 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62; and        -   ii) said second antigen-binding site comprises        -   a VH domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:92 wherein the VH            domain comprises (a) a CDR-H1 comprising the amino acid            sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the            amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77            wherein X is D or E, and (c) a CDR-H3 comprising the amino            acid sequences GVFGFFXH of SEQ ID NO:78 wherein X is D or E;        -   and a VL domain sequence having at least 90%, 91%, 92%, 93%,            94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to            the amino acid sequence of SEQ ID NO:93 wherein the VL            domain comprises (d) a CDR-L1 comprising the amino acid            sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F or T            and X² is R or L, (e) a CDR-L2 comprising the amino acid            sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   12. The bispecific antibody according to anyone of the embodiments 8    to 11, wherein the bispecific antibody a Fc domain of human IgG    isotype, preferably of human IgG1 isotype.-   13. The bispecific antibody according to anyone of the embodiments 8    to 11, wherein the bispecific wherein the bispecific antibody    comprises a constant domain of human IgG isotype, preferably of    human IgG1 isotype.-   14. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody    -   i) blocks binding of CCL2 to its receptor CCR2 in vitro        (reporter assay, IC₅₀=0.5 nM); and/or    -   ii) inhibits CCL2-mediated chemotaxis of myeloid cells in vitro        (IC₅₀=1.5 nM); and/or    -   iii) is cross-reactive to cyno and human CCL2.-   15. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody is not cross-reactive    to other CCL homologs, (shows 100 time less binding to other CCL    homologs (selected from the group of CCL8, CCL7, and CCL13) compared    to the binding to CCL2-   16. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody binds to the first and    second epitope on human CCL2 in ion-dependent manner.-   17. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody binds to human CCL2 in    pH dependent manner and wherein the first antigen binding site and    the second antigen binding site both bind to CCL2 with a higher    affinity at neutral pH than at acidic pH.-   18. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody binds to human CCL2    with a 10 times higher affinity at pH 7.4, than at pH 5.8.-   19. The bispecific antibody according to anyone of the preceding    claims, wherein the bispecific antibody, comprises a human IgG1    heavy chain constant domain comprising one or more of the following    mutations (Kabat EU numbering)    -   i) Q311R and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L234Y, L235W, G236N, P238D, T250V, V264I, T307PH268D, Q295L,        K326T and/or A330K (suitable for increasing affinity to human        FcgRIIb and decreasing affinity to other human FcgR); and/or    -   iii) M428L, N434A and/or Y436T (suitable for increasing affinity        to FcRn for longer plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).-   20. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody, comprises a human IgG1    heavy chain constant domain comprising one or more of the following    mutations (Kabat EU numbering)    -   i) Q311R, and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L235W, G236N, H268D, Q295L, K326T and/or A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and/or    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).-   21. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody, comprises a human IgG1    heavy chain constant domain comprising the following mutations    (Kabat EU numbering)    -   i) Q311R and P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L235W, G236N, H268D, Q295L, K326T and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).-   22. The bispecific antibody according to anyone of the preceding    claims, wherein the bispecific antibody, comprises a human IgG1    heavy chain constant domain comprising one or more of the following    mutations (Kabat EU numbering)    -   i) Q311R and/or P343R (suitable for increasing pI for enhancing        uptake of antigen); and/or    -   ii) L234Y, P238D, T250V, V264I, T307V and/or A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and/or    -   iii) M428L, N434A and/or Y436T (suitable for increasing affinity        to FcRn for longer plasma half-life); and/or    -   iv) Q438R and/or S440E (suitable for suppressing rheumatoid        factor binding).-   23. The bispecific antibody according to anyone of the preceding    claims, wherein the bispecific antibody, comprises a human IgG1    heavy chain constant domain comprising one or more of the following    mutations (Kabat EU numbering)    -   i) Q311R and/P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) M428L, N434A and Y436T (suitable for increasing affinity to        FcRn for longer plasma half-life); and/    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).-   24. The bispecific antibody according to anyone of the preceding    claims, wherein the bispecific antibody, comprises a human IgG1    heavy chain constant domain comprising one or more of the following    mutations (Kabat EU numbering)    -   i) Q311R and/P343R (suitable for increasing pI for enhancing        uptake of antigen); and    -   ii) L234Y, P238D, T250V, V264I, T307V and A330K (suitable for        increasing affinity to human FcgRIIb and decreasing affinity to        other human FcgR); and    -   iii) N434A (suitable for increasing affinity to FcRn for longer        plasma half-life); and/    -   iv) Q438R and S440E (suitable for suppressing rheumatoid factor        binding).-   25. The bispecific antibody according to anyone of the preceding    embodiments, wherein the bispecific antibody comprises two human    IgG1 heavy chain constant domains comprising the following mutations    (EU numbering)    -   i) S354C and T366W in one of the heavy chain constant domains    -   ii) Y349C, T366S, L368A, Y407V in the other of the heavy chain        constant domains-   26. An (isolated) antibody that (specifically) binds to a human    CCL2, wherein the antibody comprises    -   A) a VH domain comprising (a) a CDR-H1 comprising the amino acid        sequence SHYGXS of SEQ ID NO: 57 wherein X is I or T, (b) a        CDR-H2 comprising the amino acid sequence GX¹IX²IFX³TANYAQKFQG        of SEQ ID NO: 58 wherein X¹ is V, I, or H, X² is P or H, and X³        is H or G, and (c) a CDR-H3 comprising the amino acid sequence        YDAHYGELDF of SEQ ID NO: 59;        -   and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence RASQHVSDAYLA of SEQ ID NO: 60; (e) a CDR-L2            comprising the amino acid sequence DASDRAE of SEQ ID NO: 61,            and (f) a CDR-L3 comprising the amino acid sequence            HQYIHLHSFT of SEQ ID NO: 62;        -   or    -   B) a VH domain comprising (a) a CDR-H1 comprising the amino acid        sequence HTYMH of SEQ ID NO: 76, (b) a CDR-H2 comprising the        amino acid sequence RIDPXNHNTKFDPKFQG of SEQ ID NO: 77 wherein X        is D or E, and (c) a CDR-H3 comprising the amino acid sequences        GVFGFFXH of SEQ ID NO:78 wherein X is D or E; and        -   a VL domain comprising (d) a CDR-L1 comprising the amino            acid sequence KAX¹EDIYNRX²A of SEQ ID NO: 79 wherein X¹ is F            or T and X² is R or L, (e) a CDR-L2 comprising the amino            acid sequence GATSLEH of SEQ ID NO: 80, and (f) a CDR-L3            comprising the amino acid sequence QQFXSAPYT of SEQ ID NO:            81 wherein X is W or R.-   27. An (isolated) antibody that (specifically) binds to a human    CCL2, wherein the antibody comprises    -   A) a VH domain comprising the amino acid sequence of SEQ ID        NO:71;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   B) a VH domain comprising the amino acid sequence of SEQ ID        NO:72;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   C) a VH domain comprising the amino acid sequence of SEQ ID        NO:73;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   D) a VH domain comprising the amino acid sequence of SEQ ID        NO:74;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:75;    -   or    -   E) a VH domain comprising the amino acid sequence of SEQ ID        NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   F) a VH domain comprising the amino acid sequence of SEQ ID        NO:91;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:93;    -   or    -   G) a VH domain comprising the amino acid sequence of SEQ ID        NO:92;        -   and a VL domain comprising the amino acid sequence of SEQ ID            or    -   H) a VH domain comprising the amino acid sequence of SEQ ID        NO:90;        -   and a VL domain comprising the amino acid sequence of SEQ ID            NO:94;-   28. Isolated nucleic acid encoding the antibody according to any one    of the preceding embodiments.-   29. A host cell comprising the nucleic acid of embodiment 28.-   30. A method of producing an antibody comprising culturing the host    cell of embodiment 29 so that the antibody is produced.-   31. The method of embodiment 30, further comprising recovering the    antibody from the host cell.-   32. A pharmaceutical formulation comprising the bispecific antibody    according any one of embodiments 1 to 25 and a pharmaceutically    acceptable carrier.-   33. The bispecific antibody according any one of embodiments 1 to 25    for use as a medicament.-   34. The bispecific antibody according any one of embodiments 1 to 25    for use in treating cancer.-   35. The bispecific antibody according any one of embodiments 1 to 25    for use in treating an inflammatory or autoimmune disease.-   36. Use of the bispecific antibody according any one of embodiments    1 to 25 in the manufacture of a medicament.-   37. The use of embodiment 36, wherein the medicament is for    treatment of cancer.-   38. The use of embodiment 36, wherein the medicament is for    treatment of an inflammatory or autoimmune disease.-   39. A method of treating an individual having cancer comprising    administering to the individual an effective amount of the    bispecific antibody according any one of embodiments 1 to 25.-   40. A method of treating an individual having an inflammatory or    autoimmune disease comprising administering to the individual an    effective amount of the bispecific antibody according any one of    embodiments 1 to 25.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Description of the Amino Acid Sequences Anti-CCL2 Antigen BindingMoieties (Variable Regions and Hypervariable Regions (CDRs)) Binding toDifferent Epitopes:

SEQ  1 heavy 1A4 WIC ID chain NO: CDR-H1 SEQ  2 heavy 1A4CIGAGSSGSTYYASWAKG ID chain NO: CDR-H2 SEQ  3 heavy 1A4 TGTEFTYYSL IDchain NO: CDR-H3 SEQ  4 light chain 1A4 QASQSVYNNNMA ID CDR-L1 NO: SEQ 5 light chain 1A4 TASSLAS ID CDR-L2 NO: SEQ  6 light chain 1A4AGYKSYSNDEYG ID CDR-L3 NO: SEQ  7 heavy 1A4 QSLEESGGDLVKPGASLTLTCTASELDFID chain YWICWVRQAPGKGLEWIACIGAGSSGS NO: variableTYYASWAKGRFTVSKTSSTTVTLQMTS domain LTAADTATYFCARTGTEFTYYSLWGPG VHTLVTVSS SEQ  8 light chain 1A4 ALVMTQTPSSVSAAVGGTVTINCQASQ ID variableSVYNNNMAWYQQKPGQPPKLLIYTAS NO: domain SLASGVPSHFRGSGSGTQFTLTISDLES VLDDAATYYCAGYKSYSNDEYGFGGGTE VVVK SEQ  9 heavy 1A5 TSYWMC ID chain NO:CDR-H1 SEQ 10 heavy 1A5 CISSSIGVTYYASWAEG ID chain NO: CDR-H2 SEQ 11heavy 1A5 TTDDNWNVGFNL ID chain NO: CDR-H3 SEQ 12 light chain 1A5QASQSIGNRYLS ID CDR-L1 NO: SEQ 13 light chain 1A5 GTSTLAS ID CDR-L2 NO:SEQ 14 light chain 1A5 QQGATISYLDNA ID CDR-L3 NO: SEQ 15 heavy 1A5QEQLVESGGDLVKPEGSLTLTCTASGF ID chain SFSTSYWMCWVRQAPGKGLELIACISS NO:variable SIGVTYYASWAEGRFTISKTSSTTVTLQ domain MTSLTVADTATYFCARTTDDNWNVGFVH NLWGPGTLVTVSS SEQ 16 light chain 1A5 AYDMTQTPASVEVGVGGTVTIKCQAS IDvariable QSIGNRYLSWYQQKPGQPPKLLIYGTS NO: domainTLASGVSSRFKGSGSGTQFTLTISGVES VL ADSATYYCQQGATISYLDNAFGGGTEV WK SEQ 17heavy 1G9 LYSYMC ID chain NO: CDR-H1 SEQ 18 heavy 1G9 CVDAGASGSTYYASWAKGID chain NO: CDR-H2 SEQ 19 heavy 1G9 GILYYTWPYPAGAIDAFDS ID chain NO:CDR-H3 SEQ 20 light chain 1G9 QASESISNYLS ID CDR-L1 NO: SEQ 21light chain 1G9 KASTLAS ID CDR-L2 NO: SEQ 22 light chain 1G9QQSYSSSNVFNT ID CDR-L3 NO: SEQ 23 heavy 1G9 QSLEESGGDLVKPGASLTLTCKASGIDFID chain SLYSYMCWVRQAPGKGLEWIACVDAG NO: variableASGSTYYASWAKGRFTISKTSSTTVTL domain QMTSLTAADTATYFCARGILYYTWPYP VHAGAIDAFDSWGPGTLVTVSS SEQ 24 light chain 1G9 AYDMTQTPASVSEPVGGTVTIKCQASEID variable SISNYLSWYQQKPGQPPKLLIYKASTLA NO: domainSGVPSRFKGSGSGTEYTVTISGVQSDD VL AATYYCQQSYSSSNVFNTFGGGTEVV VK SEQ 25heavy 2F6 NNYYMC ID chain NO: CDR-H1 SEQ 26 heavy 2F6 CISTDDSNTHYASWAQGID chain NO: CDR-H2 SEQ 27 heavy 2F6 DAHFTSYGYGFDL ID chain NO: CDR-H3SEQ 28 light chain 2F6 RASEDIENLVA ID CDR-L1 NO: SEQ 29 light chain 2F6QASKLAS ID CDR-L2 NO: SEQ 30 light chain 2F6 QGDYGSGWIMYS ID CDR-L3 NO:SEQ 31 heavy 2F6 QSLEESGGGLVQPEGSLTLTCTASGFSF ID chainNNNYYMCWVRQAPGKGLEWIGCISTD NO: variable DSNTHYASWAQGRFTISKASSTALTLQdomain VAGLTVADMATYFCARDAHFTSYGYG VH FDLWGPGTLVTVSS SEQ 32 light chain2F6 DIVMTQTPASVSAAVGGTVSINCRASE ID variable DIENLVAWYQQKPGQPPKLLIYQASKLNO: domain ASGVPSRFKGSGSGAEFTLTIGDLESAD VL AATYYCQGDYGSGWIMYSFGGGTDLV VKSEQ 33 heavy CNTO888 SYGIS ID chain NO: CDR-H1 SEQ 34 heavy CNTO888GIIPIFGTANYAQKFQG ID chain NO: CDR-H2 SEQ 35 heavy CNTO888 YDGIYGELDF IDchain NO: CDR-H3 SEQ 36 light chain CNTO888 RASQSVSDAYLA ID CDR-L1 NO:SEQ 37 light chain CNTO888 DASSRAT ID CDR-L2 NO: SEQ 38 light chainCNTO888 HQYIQLHSFT ID CDR-L3 NO: SEQ 39 heavy CNTO888QVQLVQSGAEVKKPGSSVKVSCKASG ID chain GTFSSYGISWVRQAPGQGLEWMGGIIPI NO:variable FGTANYAQKFQGRVTITADESTSTAYM domain ELSSLRSEDTAVYYCARYDGIYGELDFVH WGQGTLVTVSS SEQ 40 light chain CNTO888 EIVLTQSPATLSLSPGERATLSCRASQSID variable VSDAYLAWYQQKPGQAPRLLIYDASS NO: domainRATGVPARFSGSGSGTDFTLTISSLEPE VL DFAVYYCHQYIQLHSFTFGQGTKVEIK SEQ 41 heavyHumanized DTYMH ID chain 11K2 NO: CDR-H1 (=11K2) SEQ 42 heavy HumanizedRIDPANGNTKFDPKFQG ID chain 11K2 NO: CDR-H2 (=11K2) SEQ 43 heavyHumanized GVFGFFDY ID chain 11K2 NO: CDR-H3 (=11K2) SEQ 44 light chainHumanized KATEDIYNRLA ID CDR-L1 11K2 NO: (=11K2) SEQ 45 light chainHumanized GATSLET ID CDR-L2 11K2 NO: (=11K2) SEQ 46 light chainHumanized QQFWSAPYT ID CDR-L3 11K2 NO: (=11K2) SEQ 47 heavy HumanizedQVQLVQSGAEVKKPGSSVKVSCKASG ID chain 11K2 LTISDTYMHWVRQAPGQGLEWMGRID NO:variable (=11K2) PANGNTKFDPKFQGRVTITADTSTSTA domainYMELSSLRSEDTAVYYCARGVFGFFDY VH WGQGTTVTVSS SEQ 48 light chain HumanizedDIQMTQSPSSLSASVGDRVTITCKATED ID variable 11K2IYNRLAWYQQKPGKAPKLLISGATSLE NO: domain (=11K2)TGVPSRFSGSGSGTDYTLTISSLQPEDF VL ATYYCQQFWSAPYTFGGGTKVEIK SEQ 49 heavyABN912 HYWMS ID chain NO: CDR-H1 SEQ 50 heavy ABN912 NIEQDGSEKYYVDSVKGID chain NO: CDR-H2 SEQ 51 heavy ABN912 DLEGLHGDGYFDL ID chain NO:CDR-H3 SEQ 52 light chain ABN912 RASQGVSSALA ID CDR-L1 NO: SEQ 53light chain ABN912 DASSLES ID CDR-L2 NO: SEQ 54 light chain ABN912QQFNSYPLT ID CDR-L3 NO: SEQ 55 heavy ABN912 EVQLVQSGGGLVQPGGSLRLSCAASGFID chain TFSHYWMSWVRQAPGKGLEWLANIEQ NO: variableDGSEKYYVDSVKGRFTISRDNAKNSLY domain LQMNSLRAEDTAVYFCARDLEGLHGD VHGYFDLWGRGTLVTVSS SEQ 56 light chain ABN912 AIQLTQSPSSLSASVGDRVILTCRASQGID variable VSSALAWYQQKPGKAPKLLIYDASSLE NO: domainSGVPSRFSGSGSGPDFTLTISSLQPEDFA VL TYFCQQFNSYPLTFGGGTKVEIKCDR Modified Anti-CCL2 Antigen Binding Moieties (Variable Regions andHypervariable Regions (CDRs)):

Modified CNTO888 SEQ 57 heavy mutated SHYGXS wherein X is I or T IDchain variant NO: CDR-H1 CNTO888 SEQ 58 heavy mutatedGX¹IX²IFX³TANYAQKFQG wherein ID chain variantX¹ is V, I, or H, X² is P or H, and X³ is H NO: CDR-H2 CNTO888 or G SEQ59 heavy mutated YDAHYGELDF ID chain variant NO: CDR-H3 CNTO888 SEQ 60light chain mutated RASQHVSDAYLA ID CDR-L1 variant NO: CNTO888 SEQ 61light chain mutated DASDRAE ID CDR-L2 variant NO: CNTO888 SEQ 62light chain mutated HQYIHLHSFT ID CDR-L3 variant NO: CNTO888 SEQ 63heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain FR- variant SGGTF NO: H1CNTO888 SEQ 64 heavy mutated WVRQAPGQGLEWMG ID chain FR- variant NO: H2CNTO888 SEQ 65 heavy mutated RVTITADESTSTAYMELSSLRSEDT ID chain FR-variant AVYYCAR NO: H3 CNTO888 SEQ 66 heavy mutated WGQGTLVTVSS IDchain FR- variant NO: H4 CNTO888 SEQ 67 light chain mutatedEIVLTQSPATLSLSPGERATLSC ID FR-L1 variant NO: CNTO888 SEQ 68 light chainmutated WYQQKPGQAPRLLIY ID FR-L2 variant NO: CNTO888 SEQ 69 light chainmutated GVPARFSGSGSGTDFTLTISSLEPED ID FR-L3 variant FAVYYC NO: CNTO888SEQ 70 light chain mutated GQGTKVEIK ID FR-L4 variant NO: CNTO888 SEQ 71heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGGTFSHYGISWVRQAPGQGLEW NO: variable CNTO888 MGGVIPIFHTANYAQKFQGRVTITAdomain H0695 DESTSTAYMELSSLRSEDTAVYYC VH ARYDAHYGELDFWGQGTLVTVSS SEQ 72heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGGTFSHYGISWVRQAPGQGLEW NO: variable CNTO888 MGGIIHIFHTANYAQKFQGRVTITAdomain H0625 DESTSTAYMELSSLRSEDTAVYYC VH ARYDAHYGELDFWGQGTLVTVSS SEQ 73heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGGTFSHYGTSWVRQAPGQGLEW NO: variable CNTO888 MGGIIHIFGTANYAQKFQGRVTITAdomain H0634 DESTSTAYMELSSLRSEDTAVYYC VH ARYDAHYGELDFWGQGTLVTVSS SEQ 74heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGGTFSHYGISWVRQAPGQGLEW NO: variable CNTO888 MGGHIHIFGTANYAQKFQGRVTITdomain H0635 ADESTSTAYMELSSLRSEDTAVYY VH CARYDAHYGELDFWGQGTLVTVS S SEQ75 light chain mutated EIVLTQSPATLSLSPGERATLSCRAS ID variable variantQHVSDAYLAWYQQKPGQAPRLLI NO: domain CNTO888 YDASDRAEGVPARFSGSGSGTDFT VLL0616 LTISSLEPEDFAVYYCHQYIHLHSFT FGQGTKVEIKModified Humanized 11K2

SEQ 76 heavy mutated HTYMH ID chain variant NO: CDR-H1 humanized 11K2SEQ 77 heavy mutated RIDPXNHNTKFDPKFQG wherein X ID chain variantis D or E NO: CDR-H2 humanized 11K2 SEQ 78 heavy mutatedGVFGFFXH wherein X is D or E ID chain variant NO: CDR-H3 humanized 11K2SEQ 79 light chain mutated KAX¹EDIYNRX²A wherein X¹ is F or T ID CDR-L1variant and X² is R or L NO: humanized 11K2 SEQ 80 light chain mutatedGATSLEH ID CDR-L2 variant NO: humanized 11K2 SEQ 81 light chain mutatedQQFXSAPYT wherein X is W or R ID CDR-L3 variant NO: humanized 11K2 SEQ82 heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain FR- variant SGLTISNO: H1 humanized 11K2 SEQ 83 heavy mutated WVRQAPGQGLEWMG ID chain FR-variant NO: H2 humanized 11K2 SEQ 84 heavy mutatedRVTITADTSTSTAYMELSSLRSEDT ID chain FR- variant AVYYCAR NO: H3 humanized11K2 SEQ 85 heavy mutated WGQGTTVTVSS ID chain FR- variant NO: H4humanized 11K2 SEQ 86 light chain mutated DIQMTQSPSSLSASVGDRVTITC IDFR-L1 variant NO: 11K2 SEQ 87 light chain mutated WYQQKPGKAPKLLIH IDFR-L2 variant NO: humanized 11K2 SEQ 88 light chain mutatedGVPSRFSGSGSGTDYTLTISSLQPED ID FR-L3 variant FATYYC NO: humanized 11K2SEQ 89 light chain mutated FGGGTKVEIK ID FR-L4 variant NO: humanized11K2 SEQ 90 heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGLTISHTYMHWVRQAPGQGLEW NO: variable humanized MGRIDPDNHNTKFDPKFQGRVTITdomain 11K2 ADTSTSTAYMELSSLRSEDTAVYY VH H1503 CARGVFGFFDHWGQGTTVTVSS SEQ91 heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGLTISHTYMHWVRQAPGQGLEW NO: variable humanized MGRIDPDNHNTKFDPKFQGRVTITdomain 11K2 ADTSTSTAYMELSSLRSEDTAVYY VH H1510 CARGVFGFFEHWGQGTTVTVSS SEQ92 heavy mutated QVQLVQSGAEVKKPGSSVKVSCKA ID chain variantSGLTISHTYMHWVRQAPGQGLEW NO: variable humanized MGRIDPENHNTKFDPKFQGRVTITAdomain 11K2 DTSTSTAYMELSSLRSEDTAVYYC VH H1514 ARGVFGFFEHWGQGTTVTVSS SEQ93 light chain mutated DIQMTQSPSSLSASVGDRVTITCKA ID variable variantFEDIYNRRAWYQQKPGKAPKLLIH NO: domain humanized GATSLEHGVPSRFSGSGSGTDYTLTVL 11K2 ISSLQPEDFATYYCQQFWSAPYTFG L1338 GGTKVEIK SEQ 94 light chainmutated DIQMTQSPSSLSASVGDRVTITCKA ID variable variantTEDIYNRLAWYQQKPGKAPKLLIH NO: domain humanized GATSLEHGVPSRFSGSGSGTDYTLTVL 11K2 ISSLQPEDFATYYCQQFRSAPYTFG L1201 GGTKVEIKExemplary Constant Light Chain Regions:

-   SEQ ID NO: 95 exemplary human kappa light chain constant region-   SEQ ID NO: 96 exemplary human lambda light chain constant region    Exemplary Constant Heavy Chain Regions:-   SEQ ID NO: 97 exemplary human heavy chain constant region derived    from IgG1-   SEQ ID NO: 98 exemplary human heavy chain constant region derived    from IgG1 with mutations L234A, L235A and P329G (Fcgamma receptor    silenced)-   SEQ ID NO: 99 exemplary human heavy chain constant region derived    from IgG1 (SG1-IgG1 allotype) SEQ ID NO: 100 exemplary human heavy    chain constant region derived from IgG1 with mutations (SG105-IgG1    allotype—Fcgamma receptor silenced)-   SEQ ID NO: 101 SG1095-exemplary human heavy chain constant region    derived from IgG1 including the mutations (Kabat EU numbering):    -   L235W/G236N/H268D/Q295L/A330K/K326T (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen;    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody; and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding-   SEQ ID NO: 102 SG1099-exemplary human heavy chain constant region    derived from IgG1 including mutations (Kabat EU numbering):    -   Q311R/P343R (suitable for increasing pI for enhancing uptake of        antigen)-   SEQ ID NO: 103 SG1100-exemplary human heavy chain constant region    derived from IgG1 including the mutations (Kabat EU numbering):    -   Q311R/P343R (suitable for increasing pI for enhancing uptake of        antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)-   CNTO888/11K2-WT IgG1 (exemplary bispecific CNTO888/11K2-WT IgG1    Crossmab)-   SEQ ID NO: 104 heavy chain 1-CNTO888/11K2-WT IgG1-   SEQ ID NO: 105 heavy chain 2-CNTO888/11K2-WT IgG1-   SEQ ID NO: 106 light chain 1-CNTO888/11K2-WT IgG1-   SEQ ID NO: 107 light chain 2-CNTO888/11K2-WT IgG1-   CKLO2-IgG1 (exemplary bispecific CKLO2 IgG1 Crossmab)-   SEQ ID NO: 108 heavy chain 1-CKLO2 IgG1-   SEQ ID NO: 109 heavy chain 2-CKLO2 IgG1-   SEQ ID NO: 110 light chain 1-CKLO2 IgG1-   SEQ ID NO: 111 light chain 2-CKLO2 IgG1-   CKLO2-SG1095 (exemplary bispecific CLOK2 Crossmab including SG1095    Fc mutations)-   SEQ ID NO: 112 heavy chain 1-CKLO2-SG1095-   SEQ ID NO: 113 heavy chain 2-CKLO2-SG1095-   SEQ ID NO: 114 light chain 1-CKLO2-SG1095-   SEQ ID NO: 115 light chain 2-CKLO2-SG1095-   CKLO2-SG1099 (exemplary bispecific CKLO2 Crossmab including SG1099    Fc mutations)-   SEQ ID NO: 116 heavy chain 1-CKLO2-SG1099-   SEQ ID NO: 117 heavy chain 2-CKLO2-SG1099-   SEQ ID NO: 118 light chain 1-CKLO2-SG1099-   SEQ ID NO: 119 light chain 2-CKLO2-SG1099-   CKLO2-SG1100 (exemplary bispecific CKLO2 Crossmab including SG1100    Fc mutations)-   SEQ ID NO: 120 heavy chain 1-CKLO2-SG1100-   SEQ ID NO: 121 heavy chain 2-CKLO2-SG1100-   SEQ ID NO: 122 light chain 1-CKLO2-SG1100-   SEQ ID NO: 123 light chain 2-CKLO2-SG1100-   CKLO3-SG1095 (exemplary bispecific CLOK3 Crossmab including SG1095    Fc mutations)-   SEQ ID NO: 124 heavy chain 1-CKLO3-SG1095-   SEQ ID NO: 125 heavy chain 2-CKLO3-SG1095-   SEQ ID NO: 126 light chain 1-CKLO3-SG1095-   SEQ ID NO: 127 light chain 2-CKLO3-SG1095-   CKLO3-SG1099 (exemplary bispecific CKLO3 Crossmab including SG1099    Fc mutations)-   SEQ ID NO: 128 heavy chain 1-CKLO3-SG1099-   SEQ ID NO: 129 heavy chain 2-CKLO3-SG1099-   SEQ ID NO: 130 light chain 1-CKLO3-SG1099-   SEQ ID NO: 131 light chain 2-CKLO3-SG1099-   CKLO3-SG1100 (exemplary bispecific CKLO3 Crossmab including SG1100    Fc mutations)-   SEQ ID NO: 132 heavy chain 1-CKLO3-SG1100-   SEQ ID NO: 133 heavy chain 2-CKLO3-SG1100-   SEQ ID NO: 134 light chain 1-CKLO3-SG1100-   SEQ ID NO: 135 light chain 2-CKLO3-SG1100    Further Anti-CCL2 Antigen Binding Moieties:-   SEQ ID NO: 136 heavy chain variable domain VH 2F2-   SEQ ID NO: 137 light chain variable domain VL 2F2-   SEQ ID NO: 138 heavy chain variable domain VH murine 11K2 (=11K2m)-   SEQ ID NO: 139 light chain variable domain VL murine 11K2 (=11K2m)-   SEQ ID NO: 140 heavy chain variable domain VH 1H11-   SEQ ID NO: 141 light chain variable domain VL 1H11    Exemplary CCL2 and Homologs (without Signal Peptide):-   SEQ ID NO: 142 exemplary human CCL2 (MCP1)-wild type (wt)-   SEQ ID NO: 143 exemplary human CCL2 (MCP1)-P8A variant-   SEQ ID NO: 144 exemplary human CCL2 (MCP1)-T10C variant-   SEQ ID NO: 145 exemplary human CCL8 (MCP2)-wild type (wt)-   SEQ ID NO: 146 exemplary human CCL8 (MCP2)-P8A variant-   SEQ ID NO: 147 exemplary human CCL7 (MCP3)-wild type (wt)-   SEQ ID NO: 148 exemplary human CCL13 (MCP4)-wild type (wt)-   SEQ ID NO: 149 exemplary cynomolgus CCL2-wild type (wt)-   SEQ ID NO: 150 exemplary mouse CCL2-wild type (wt)    Further Exemplary Constant Heavy Chain Regions:-   SEQ ID NO: 151 GG01—exemplary human heavy chain constant region    derived from IgG1 including the mutations (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)-   SEQ ID NO: 152 GG02—exemplary human heavy chain constant region    derived from IgG1 including mutations (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   M428L/N434A/Y436T (suitable for increasing affinity to FcRn for        longer plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)-   SEQ ID NO: 153 GG03—exemplary human heavy chain constant region    derived from IgG1 (comprising-IgG1 allotype sequences) including the    mutations (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)-   SEQ ID NO: 154 GG04—exemplary human heavy chain constant region    derived from IgG1 (comprising-IgG1 allotype sequences) including    mutations (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   M428L/N434A/Y436T (suitable for increasing affinity to FcRn for        longer plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)-   CKLO2-GG01 (exemplary bispecific CLOK2 Crossmab including GG01 Fc    mutations)-   SEQ ID NO: 155 heavy chain 1-CKLO2-GG01-   SEQ ID NO: 156 heavy chain 2-CKLO2-GG01-   SEQ ID NO: 157 light chain 1-CKLO2-GG01-   SEQ ID NO: 158 light chain 2-CKLO2-GG01-   CKLO2-GG02 (exemplary bispecific CLOK2 Crossmab including GG02 Fc    mutations)-   SEQ ID NO: 159 heavy chain 1-CKLO2 GG02-   SEQ ID NO: 160 heavy chain 2-CKLO2 GG02-   SEQ ID NO: 161 light chain 1-CKLO2 GG02-   SEQ ID NO: 162 light chain 2-CKLO2 GG02-   CKLO2-GG03 (exemplary bispecific CLOK2 Crossmab including GG03 Fc    mutations)-   SEQ ID NO: 163 heavy chain 1-CKLO2-GG03-   SEQ ID NO: 164 heavy chain 2-CKLO2-GG03-   SEQ ID NO: 165 light chain 1-CKLO2-GG03-   SEQ ID NO: 166 light chain 2-CKLO2-GG03-   CKLO2-GG04 (exemplary bispecific CKLO2 Crossmab including GG04 Fc    mutations)-   SEQ ID NO: 167 heavy chain 1-CKLO2-GG04-   SEQ ID NO: 168 heavy chain 2-CKLO2-GG04-   SEQ ID NO: 169 light chain 1-CKLO2-GG04-   SEQ ID NO: 170 light chain 2-CKLO2-GG04-   CKLO2-GG03/GG04 (exemplary bispecific CKLO2 Crossmab including GG03    Fc mutations in the Knob chain and including GG04 Fc mutations in    Hole chain)-   SEQ ID NO: 171 heavy chain 1-CKLO2-GG03/GG04-   SEQ ID NO: 172 heavy chain 2-CKLO2-GG03/GG04-   SEQ ID NO: 173 light chain 1-CKLO2-GG03/GG04-   SEQ ID NO: 174 light chain 2-CKLO2-GG03/GG04    Designation Monospecific Unmodified Anti-CCL2 Antibodies/Antigen    Binding Moieties which were Used for the Anti-CCL2 Bispecific    Antibodies Described Herein

Antibody/antigen binding site Alias VH/VL 1A4 CCL2-0008 SEQ ID NO: 8/SEQID NO: 9 1A5 CCL2-0009 SEQ ID NO: 15/SEQ ID NO: 16 1G9 CCL2-0010 SEQ IDNO: 23/SEQ ID NO: 24 2F6 CCL2-0014 SEQ ID NO: 31/SEQ ID NO: 32 CNTO888CCL2-0004 SEQ ID NO: 39/SEQ ID NO: 40 Humanized CCL2-0002 SEQ ID NO:47/SEQ ID NO: 48 11K2 (=11K2) ABN912 CCL2-0003 SEQ ID NO: 55/SEQ ID NO:56Designation Bispecific Anti-CCL2 with Unmodified VH/VL as Crossmabs (SeeWO 2016/016299) with Either IgG1 or IgG1 Including Mutations L234A,L235A and P329G (PGLALA)

Bispecific anti-CCL2 Antibodies Alias 11K2//1G9-WT IgG1 CCL2-004911K2//1G9-PGLALA CCL2-0043 CNTO888//11K2-WT IgG1 CCL2-0048CNTO888//11K2-PGLALA CCL2-0042 CNTO888//1G9-WT IgG1 CCL2-0051CNTO888//1G9-PGLALA CCL2-0045 CNTO888//1A5-WT IgG1 CCL2-0050CNTO888//1A5-PGLALA CCL2-0044 1A5//1G9-WT IgG1 CCL2-0052 1A5//1G9-PGLALACCL2-0046 11K2//2F6-WT IgG1 CCL2-0056 11K2//2F6-PGLALA CCL2-0053ABN912//11K2-WT IgG1 CCL2-0047 ABN912//11K2-PGLALA CCL2-0041 1A4//2F6-WTIgG1 CCL2-0057 1A4//2F6-PGLALA CCL2-0054 1A5//2F6-WT IgG1 CCL2-00581A5//2F6-PGLALA CCL2-0055Designation Bispecific Antibodies with Modified VH/VL as Crossmabs (SeeWO 2016/016299). Depending on the Heavy Chain Constant Domain Used (e.g.IgG1 Wild Type, PGLALA, SG1095, SG1099, 1100) the Suffixes IgG1 WildType, PGLALA, SG1095, SG1099, 1100 are Added

Bispecific CCL2 VH VL VH VL antibody (and (SEQ (SEQ (SEQ (SEQ parentalName of variable region ID ID ID ID monospecific) (VHs/VLs) NO) NO) NO)NO) Humanized 11K2 VH/11K2 VL 47 48 — — 11K2 parental CNTO888 CNTO888VH/ 39 40 parental CNTO888VL CKLO01 11K2H1503/11K2L1338// 90 93 71 75CNTO888H0695/CNTO8 88L0616 CKLO02 11K2H1510/11K2L1338// 91 93 71 75CNTO888H0695/CNTO8 88L0616 CKLO03 11K2H1503/11K2L1201// 90 94 71 75CNTO888H0695/CNTO8 88L0616 CKLO04 11K2H1503/11K2L1201// 90 94 72 75CNTO888H0625/CNTO8 88L0616 CKLO05 11K2H1503/11K2L1338// 90 93 73 75CNTO888H0634/CNTO8 88L0616 CKLO06 11K2H1503/11K2L1201// 90 94 73 75CNTO888H0634/CNTO8 88L0616 CKLO07 11K2H1514/11K2L1338// 92 93 73 75CNTO888H0634/CNTO8 88L0616 CKLO08 11K2H1510/11K2L1338// 91 93 73 75CNTO888H0634/CNTO8 88L0616 CKLO09 11K2H1503/11K2L1338// 90 93 72 75CNTO888H0625/CNTO8 88L0616 CKLO10 11K2H1514/11K2L1338// 92 93 72 75CNTO888H0625/CNTO8 88L0616 CKLO11 11K2H1510/11K2L1338// 91 93 72 75CNTO888H0625/CNTO8 88L0616 CKLO12 11K2H1503/11K2L1338// 90 93 74 75CNTO888H0635/CNTO8 88L0616 CKLO13 11K2H1503/11K2L1201// 90 94 74 75CNTO888H0635/CNTO8 88L0616 CKLO14 11K2H1514/11K2L1338// 92 93 74 75CNTO888H0635/CNTO8 88L0616 CKLO15 11K2H1510/11K2L1338// 91 93 74 75CNTO888H0635/CNTO8 88L0616 CKLO16 11K2H1514/11K2L1338// 92 93 71 75CNTO888H0695/CNTO8 88L0616

EXAMPLES Example A-1 Monospecific Anti-CCL2 Antibodies

Generation of monospecific anti-CCL2 antibodies and CCL2 antigen

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany). The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequencesof subcloned gene fragments were verified by DNA sequencing.Alternatively, short synthetic DNA fragments were assembled by annealingchemically synthesized oligonucleotides or via PCR. The respectiveoligonucleotides were prepared by metabion GmbH (Planegg-Martinsried,Germany)

Description of the Basic/Standard Mammalian Expression Plasmid

For the expression of a desired gene/protein (e.g. full length antibodyheavy chain, full length antibody light chain, or a CCL-2 molecule, atranscription unit comprising the following functional elements is used:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a gene/protein to be expressed (e.g. full length antibody heavy        chain or antibody light chain or CCL-2 molecule), and    -   the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene to beexpressed the basic/standard mammalian expression plasmid contains

an origin of replication from the vector pUC18 which allows replicationof this plasmid in E. coli, and

a beta-lactamase gene which confers ampicillin resistance in E. coli.

Generation of Expression Plasmids for Recombinant Monoclonal Antibodiesand CCL-2 Molecules

The expression plasmids for the transient expression of monoclonalantibodies and CCL-2 antigens comprised besides the respectiveexpression cassettes an origin of replication from the vector pUC18,which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of the respective immunoglobulin HC or LC orCCL-2 molecule comprised the following functional elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence, and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        The Respective Antibodies 1A4, 1A5, 1G9, 2F6, CNTO888, Murine        and Humanized 11K2, ABN912, Based on their VH and VL were        Generated as I2G1 Wild Type and as I2G1 PGLALA/Effector Silent        Fc with Kappa Light Chain        Transient Expression and Purification

The recombinant production was performed by transient transfection ofHEK293 cells (human embryonic kidney cell line 293-derived) cultivatedin F17 Medium (Invitrogen Corp.). For the production of monoclonalantibodies, cells were co-transfected with plasmids containing therespective immunoglobulin heavy- and light chain. For transfection“293-Fectin” Transfection Reagent (Invitrogen) was used. Transfectionwas performed as specified in the manufacturer's instructions. Cellculture supernatants were harvested three to seven (3-7) days aftertransfection. Supernatants were stored at reduced temperature (e.g. −80°C.).

General information regarding the recombinant expression of humanimmunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al.,Biotechnol. Bioeng. 75 (2001) 197-203.

Antibodies were purified from cell culture supernatants by affinitychromatography using MabSelectSure-Sepharose™ (GE Healthcare, Sweden)and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.Briefly, sterile filtered cell culture supernatants were captured on aMabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mMKH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibrationbuffer and eluted with 25 mM sodium citrate at pH 3.0. The elutedprotein fractions were pooled, neutralized with 2M Tris, pH 9.0 andfurther purified by size exclusion chromatography using a Superdex 20026/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mMhistidine, 140 mM NaCl, pH 6.0. Size exclusion chromatography fractionswere analysed by CE-SDS (Caliper Life Science, USA) and antibodycontaining fractions were pooled and stored at −80° C.

Generation of Recombinant CCL2

Wild type CCL2 can exist as monomer but actually can also form dimers atphysiological concentrations. This monomer-dimer equilibrium might bedifferent and has to be carefully taken into account for all in vitroexperiments described where different concentrations might be used. Toavoid any uncertainties, we generated point mutated CCL2 variants: TheP8A variant of CCL2 carries a mutation in the dimerization interfaceresulting in an inability to form a dimer leading to a defined, pureCCL2 monomer. In contrast, the T10C variant of CCL2 results in a fixeddimer of CCL2 (J Am Chem Soc. 2013 Mar. 20; 135(11):4325-32).

The respective soluble CCL2 protein (wild type, P8A or T10C variants)was purified from cell culture supernatants by cation exchangechromatography using SP-Sepharose HP (GE Healthcare, Sweden) andSuperdex 200 size exclusion (GE Healthcare, Sweden) chromatography.Briefly, sterile filtered cell culture supernatants were diluted with 10mM KH2PO4, pH 5.0 to adjust conductivity <4 mS/cm. The dilutedsupernatant was loaded on SP-Sepharose resin equilibrated with 10 mMKH2PO4, pH 5.0, washed with equilibration buffer and eluted using agradient to 10 mM KH2PO4, 1 M NaCl, pH 5.0. The eluted protein fractionswere pooled and further purified by size exclusion chromatography usinga Superdex 200 16/60 GL (GE Healthcare, Sweden) column equilibrated with20 mM histidine, 140 mM NaCl, pH 6.0. Size exclusion chromatographyfractions were analyzed by SDS-PAGE and analytical high performance sizeexclusion chromatography. CCL2 containing fractions were pooled andstored at −80° C.

Functional Characterization (Binding)

A T200 instrument was mounted with a Biacore Series S Sensor Chip CM5.The system buffer was HBS-ET (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mMEDTA, 0.05% (w/v) P20). The system was set to 37° C. For eachmeasurement the sample buffer was the system buffer, additionallysupplemented with 1 mg/ml CMD (Carboxymethyldextran, Fluka).

An antibody capture system was established. 14000 GARFcγ (goat antirabbit Fcγ), 111-005-046, Jackson ImmunoResearch) were immobilized at25° C. at 25 μg/ml in 10 mM sodium acetate buffer pH 5.0, by EDC/NHScoupling as described by the manufacturer. The capture system wasregenerated at 20 μl/min by a 15 sec injection with HBS buffer (100 mMHEPES pH 7.4, 1.5 M NaCl, 0.05% (w/v) Tween 20), a 1 min injection with10 mM glycine buffer pH 2.0 followed by two injections for 1 min with 10mM glycine buffer pH 2.25. In another embodiment murine monoclonalantibodies were captured on the biosensor by immobilizing 12700 RUpolyclonal rabbit anti mouse (RAMIgG, GE Healthcare) antibodies on aBiacore Series CM5 sensor like described above. The sensor wasregenerated by a 3 min injection of 10 mM glycine buffer pH 1.7.

Antibody clone supernatants were diluted 1:2 in system buffer and werecaptured for 1 min at 5 μl/min. After antibody capturing the system waswashed by 2.5-fold concentrated system buffer for 30 sec at 80 μl/minfollowed by 2 min baseline stabilization. Analyte kinetics wereperformed at 30 μl/min. As analyte in solution wt human CCL2 ormonomeric CCL2 P8A variant CCL2 were used. Analytes were injected at 90nM highest concentration. The analyte contact time was 3 min and thedissociation time was 10 min. The Biaevaluation software V.3.0 was usedaccording to the instructions of the manufacturer GEHC. A 1:1 bindingmodel with RMAX local was applied to apparently estimate kinetic rates.

Binding of Antibodies to Wild Type (Wt) Human CCL2 and Human CCL2 P8AVariant (Monomer)

KD [nM] KD [nM] wt human T1/2 monomeric T1/2 Antibody alias CCL2 [min]human CCL2 [min] 1A4 CCL2-0008 0.073 100 0.059 174 1A5 CCL2-0009 0.024160 0.046 137 1G9 CCL2-0010 0.062 177 0.051 218 2F6 CCL2-0014 0.059 520.091 47 murine 11K2 X-0048 0.35 19 0.011 518 humanized CCL2-0002 0.028118 0.035 116 11K2 (=11K2) AB912 CCL2-0003 0.036 33 0.046 25 CNTO888CCL2-0004 0.026 94 0.054 44Summary pH Dependent CCL2 Binding Kinetics Obtained from SPR Analysis I

A T200 instrument was mounted with a Biacore Series S Sensor Chip CM5.The system buffer was HBS-ET (10 mM HEPES (pH 7.4), 150 mM NaCl, 1 mMEDTA, 0.05% (w/v) P20). In other embodiments the pH of the systembuffers was set to pH 8.3, pH 7.9, pH 7.4, pH 7.1, pH 6.7, pH 6.3, pH5.9, pH 5.5. The system was set to 25° C. For each measurement thesample buffer was the system buffer, additionally supplemented with 1mg/ml CMD (Carboxymethyldextran, Fluka).

An antibody capture system was established. 13000MAb<h-Fc-pan>M-R10Z8E9-IgG (Roche) were immobilized at 25° C. at 18μg/ml in 10 mM sodium acetate buffer pH 5.0, by EDC/NHS coupling asdescribed by the manufacturer. The capture system was regenerated by aninjection at 20 μl/min with HBS buffer (100 mM HEPES pH 7.4, 1.5 M NaCl,0.05% (w/v) Tween 20), followed by a 1 min 15 sec injection with 10 mMNaOH and two 10 mM glycine buffer pH 2.5 injections for 1 min.Antibodies were captured were injected for 30 sec at 10 μl/min at 80 nMconcentration diluted in the respective system buffer. After antibodycapturing the system was washed by 2.5-fold concentrated system bufferfor 30 sec at 50 μl/min followed by 2 min baseline stabilization.Concentration-dependent analyte series were injected in 1:3 dilutionsteps, from 0 nM (buffer control) 0.4 nM, 1.1 nM, two injections at 3.3nM, 30 nM. The analyte contact time was 3 min and the dissociation timewas 10 min. Analyte kinetics were performed at 50 μl/min.

Human antibodies were captured as ligands on the sensor surface:

-   -   Human normal IgG as positive control (H-N-IgG, Id.: 11717570,        Roche),    -   anti-human CCL2 mAb (humanized 11k2: CCL2-0002),    -   anti-human CCL2 mAb (AB912, CCL2-0003), and    -   anti-human CCL2 mAb (CNTO888, CCL2-0004);    -   system buffer as negative control.

The Biaevaluation software V.3.0 was used according to the instructionsof the manufacturer GEHC. A 1:1 binding model with R_(MAX) local wasapplied to determine kinetic rates.

humanized 11K2 ABN912 CNTO888 CCL2-0002 CCL2-0003 CCL2-0004 t ½- t ½- t½- KD diss. KD diss. KD diss. pH [nM] [min] [nM] [min] [nM] [min] 8.30.003 1155 0.004 35 0.01 135 7.9 0.02 165 0.004 25 0.01 138 7.4 0.02 1630.01 33 0.02 130 7.1 0.01 215 0.02 20 0.02 124 6.7 0.01 292 0.2 8 0.03103 6.3 0.003 1155 1 3 0.02 128 5.9 0.01 287 13 1 0.04 61 5.5 0.01 1182000 0.03 0.06 31

Crossreactivity CCL homologs

As CCL2 (MCP-1) has high homology to CCL7 (MCP-3), CCL8 (MCP-2), CCL13(MCP-4), and these CCL chemokines are able to bind to CCR2, the bindingof anti-CCL2 antibodies to these homologs was assessed. Results areshown in FIG. 1 , FIG. 1A, FIG. 1B, and FIG. 1C. With the exception ofCNTO888 which was described to have selectivity to CCL2 (Mol Immunol.2012 June; 51(2):227-33), the other antibodies tested bound to eitherCCL7 or CCL8 (showed cross-reactivity to either CCL7 or CCL8).

Biacore assay method: The binding of anti-CCL2 antibodies to the CCLhomologs e.g. CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3), and CCL13(MCP-4) were assessed at 25° C. using Biacore T200 instrument (GEHealthcare). Mouse anti-human IgG (Fc) (GE Healthcare) was immobilizedon each flow cells of a CM4 sensor chip using amine coupling kit (GEHealthcare) according to the recommended settings by the manufacturer.Antibodies and analytes were diluted into ACES pH 7.4 buffer (20 mMACES, 150 mM NaCl, 1 mg/ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibodieswere captured onto the anti-Fc sensor surfaces, then recombinant humanCCL homologs proteins was injected over the flow cell at 5 nM and 20 nM.Wild type CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3), and CCL13 (MCP-4)were commercially available from R&D Systems, whereas monomer CCL2 (P8Avariant) was in-house generated antigen. Sensor surface was regeneratedeach cycle with 3M MgCl2. Binding sensorgram was processed using BiacoreT200 Evaluation software, version 2.0 (GE Healthcare).

Functional Characterization (Biological)

CCR2 Signaling I—Calcium Flux Assay

THP-1 (human acute monocytic leukemia cell line; ATCC TIB-202) cellswere cultivated in RPMI 1640, 10% FBS, 1 mM sodium pyruvate, 10 mMHEPES, 50 μM ß-mercaptoethanol (supplier Thermo Fisher Scientific). Onthe assay day the cell density was adjusted to 8.33×10⁵ cells/ml in 25.8ml assay medium (RPMI 1640 w/o FBS). FLIPR® Calcium Assay Kits (FLIPRCalcium 4 Assay Kit, Cat #R8142, Molecular Devices) were used fordetecting intracellular calcium changes in a homogeneous assay format.

A dye loading solution was prepared by mixing two vials of component Awith 20 ml component B (HBSS buffer plus 20 mM HEPES, pH 7.4) accordingto the instructions of the manufacturer Molecular Devices. 516 μl 1 MHepes (final assay concentration: 10 mM) is added followed by 516 μl 250mM probenecid (final assay concentration: 2.5 mM). For the stocksolution dissolve 65.4 mg probenecid (Sigma P8761) in 465 μl 1 N NaOHand add 465 μl 1×HBSS (Thermo Fisher Scientific). 25.8 ml loading bufferwas mixed with 25.8 ml assay medium with cells sufficient for e.g. fourmicrotiter plates (52.6 ml volume is needed; 10⁶ THP-1/ml). 120 μl cellsuspension in loading buffer was transferred to each well of a blackF-bottom 96-well cell culture plate. The plates were incubated at roomtemperature for 3-4 hours.

In the meantime, the antibody and the ligand solution were prepared.Eight concentrations of each antibody from 30 μg/ml to 0.025 μg/ml (noserial dilution, final concentration in wells) have been tested. Eachconcentration was tested on two plates. All dilutions were prepared inassay medium as 10-fold concentrated solution. As reference antibodyhuman CCL2/JE/MCP-1 Antibody (R&D Systems Cat #MAB279) was used. LigandCCL2 (R&D Systems Cat #279-MC-10) was prepared by dissolving 50 μg CCL2lyophilisate in 500 μl RPMI 1640 (100 μg/ml) and transferring 400 μlinto 10 ml assay medium (4 μg/ml stock solution). As stimulation controlionomycin (Sigma Cat #I-0634) was used (1 mg ionomycin dissolved in 1340μl DMSO (Sigma Cat #D-8779), 1 mM). 10 μl of the 1 mM stock solution wasdiluted in 1990 μl assay medium (5 μM, final assay concentration 500nM). 100 μl was pipetted in the corresponding control wells of thepolypropylene MTP.

The antibody dilutions and CCL2 were preincubated in two V-shapepolypropylene 96 well plates. 50 μl of the 4 μg/ml stock solution CCL2(final 400 ng/ml CCL2) and 50 μl of the 10-fold concentrated antibodydilution were pipetted into the well. Plates were incubated for 30-60min at room temperature.

After incubation, the cell plate and the compound plate were transferreddirectly to the FlexStation® 3 (Molecular Devices) read position and thecalcium assay was performed as described in the system manual(excitation 485 nm, emission 525 nm). The read out was done at severalseconds interval.

Results:

TABLE 1 40 ng/ml PMA and 4 μM ionomycin were used as positive controls.The table includes mean of EC₅₀ of n = 2. Anti-CCL2 antibodiesInhibition of CCR2 signaling Antibody Alias EC50 [μg/ml] EC50 [nM] 1A4CCL2-0008 4.2 28.0 1A5 CCL2-0009 2.2 14.6 1G9 CCL2-0010 2.4 16 2F6CCL2-0014 8.7 3.9 Mab279 (R&D) Commercial 2.6 17.4 reference murine 11K2X-0048 4.5 30.0 (11K2m) Humanized 11K2 CCL2-0002 2.1 14.0 (11K2) AB912CCL2-0003 1.9 12.7 CNTO888 CCL2-0004 2.7 18Potency of Anti-CCL2 Antibodies to Inhibit CCL2-Induced Internalizationof the CCR2 Receptor Expressed on Monocytes

To prevent the ligand-induced CCR2 internalization on myeloid cells weset up an in vitro assay and characterized anti-CCL2 antibodies.Monocytes were isolated from peripheral blood of healthy donors bymagnetic separation using a commercial kit (Stemcell, cat no. #15068).For blocking of FcγRs, monocytes were pre-incubated with normal humanIgG (Privigen, CSL Behring) at a final concentration of 500 μg/ml on icefor 50 min in FACS buffer (PBS+0.2% BSA). Cells were then centrifugedfor 10 min (300×g, 4° C.), washed one more time with FACS buffer andstored on ice. Anti-CCL2 antibody dilutions (50 μl each) were prepared(in parallel approaches at 4° C. and 37° C.) in 96 U-bottom wells (BD).Monocytes were split, re-suspended in medium (RPMI 1640; 10% FCS; 2 mML-Glutamine) and incubated at 4° C. and 37° C., respectively, untilfurther usage. Recombinant CCL2 (50 μl; at a final concentration of 100ng/ml) was added to the prepared antibody dilutions (at variableconcentrations) both at 4° C. and 37° C. 100 μl monocyte suspension(2×10⁵ cells/well) was added to the CCL2/anti-CCL2 mixes at a totalvolume of 200 μl and cells were incubated at 4° C. and 37° C. for 1 h 30min before centrifugation at 300×g, 4° C. From now on all steps wereconducted with pre-cooled buffers: cells were washed with 250 μl FACSbuffer and additionally, cells were stained against CCR2 receptor (usinga commercial CCR2-APC conjugate or appropriate isotype ctrl-APCaccording standard FACS protocols: Aliquots were stained with 5 μl/10⁶cell with CD192 (CCR2) APC (BioLegend, #357208, clone K036C2/mIgG2a κ)as well as an appropriate isotype ctrl antibody: 20 μl/10⁶ cell mIgG2a kAPC BD Biosciences, #400222, clone MOPC-173).

Then the receptor expression was analyzed on a FACS Canto II and theCCR2 internalization was calculated as follows:

-   -   No internalization: Cells analyzed in the absence of ligand        (rec. CCL2) incubation.    -   100% internalization: Maximally reduced CCR2 expression level on        cells previously incubated with rec. CCL2

Inhibition of CCR2 Anti-CCL2 antibodies internalization Antibody AliasEC50 [μg/ml] EC50 [nM] 1A4 CCL2-0008 2.52 16.81 1A5 CCL2-0009 2.37 15.771G9 CCL2-0010 2.16 14.42 2F6 CCL2-0014 2.41 1.09 11K2 (murine) X-00481.98 13.19 AB912 CCL2-0003 1.99 13.25 CNTO888 CCL2-0004 2.00 13.36Mab279 (R&D) Commercial 1.98 13.22 referenceInhibition of CCL2-Mediated Chemotaxis on Human THP-1 Cells

The migration of CCR2+THP1 cells towards a CCL2 gradient was tested asfollows. Monocytic THP1 cells (ATCC©G TIB-202™) were cultured in RPM11640 medium (PAN, cat. no. #P04-17500) supplemented with FCS andL-Glutamine. Cells were normally passaged two to three times prior touse in the migration assay and then starved overnight in media withreduced FSC content (1.5% instead of 10% FCS). Cells were counted andincubated with 10 μg/ml normal human IgG (Invitrogen, cat. no. #12000;to block FcgRs) for 15 minutes at room temperature.

In the meantime, anti-CCL2 antibodies (and/or controls) were added tothe lower chamber of a HTS Transwell 96 well plate system (Corning, cat.no. #3386; 3 μm pore size) containing serum-free media with 25 ng/mlrhCCL-2 (R&D Systems, cat. no. #279-MC). Then the insert-plates werestuck into the lower-chamber-plate and 75 μl (1.5×10⁵ cells) of theabove mentioned cell-suspension (including the IgG-block) were addedwith or without 5 μg/ml antibody/isotype into each insert. Plates werecovered and incubated over night at 37° C. in an CO2 incubator (5% CO2).

The insert-plate was removed and Cell-titer-glo substrate (Promega, cat.no. #G758) was added to each well of lower-chamber-plate to measureviability of migrated cells. After incubation for 1 hour on a shakerwith 300 rpm (cover plate sealed), 200 μl of each well were transferredto a Microfluor black 96 well-plate (VWR, cat. no. #735-0527) andluminescence was measured (luminescence-reader e.g. Bio-Tek, Tecan).Fold change was calculated as the ratio between number of migrated cells(Cell Titer Glo, RLU) with IgG control antibody and anti-CCL2antibodies. Shown in the following Table 2 are the results of 5-10replicates per condition:

TABLE 2 THP1 chemotaxis [fold change compared to Antibody Alias IgGcontrol] 1A4 CCL2-0008 4.5 1A5 CCL2-0009 3.6 1G9 CCL2-0010 0.9 2F6CCL2-0014 6.7 Mab279 (R&D) — 3.7 Murine 11K2 (11K2m) X-0048 6.9Humanized 11K2 (11K2) CCL2-0002 7.2 ABN912 CCL2-0003 6.5 CNTO888CCL2-0004 4.9Evaluation of Human CCL2 Immune Complex Sweeping with Monospecific(Monoparatopic) Anti-CCL2 Antibodies in Mice

To evaluate the ability of monoparatopic antibodies to form immunecomplex with wild type human CCL2, pre-formed immune complexesconsisting of anti-CCL2 monoparatopic antibody (20 mg/kg) and wild typehuman CCL2 (0.1 mg/kg) were administered at a single dose of 10 ml/kginto the caudal vein of human FcRn transgenic mice(B6.Cg-Fcgrt^(tm1Dcr)Tg(FCGRT)32Dcr/DcrJ, Jackson Laboratory). Blood wascollected 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 days afteradministration. Serum was prepared by centrifuging the blood immediatelyat 14,000 rpm for 10 minutes in 4° C. The serum was stored at or below−80° C. until measurement. The monoparatopic antibodies tested arelisted in the Table 3 below. Antibodies with SG1 Fc have Fc gammareceptor binding similar to wild-type while antibodies with SG105 Fc areFc gamma receptor binding silent.

The effect of immune complex sweeping of each anti-CCL2 monoparatopicantibody on hCCL2 clearance in vivo were assessed by comparing anti-CCL2antibody with Fc gamma receptor binding (SG1, =IgG1 wild type withintact Fc gamma receptor binding; solid line) and anti-CCL2 antibodywith Fc gamma receptor binding silent (SG105, =IgG1 with no Fc gammareceptor binding; dotted line), as shown in FIG. 2 a -FIG. 2 g . Therespective FIG. 2 a -FIG. 2 g show the serum concentration of hCCL2 overtime after injection of the pre-formed immune complexes consisting ofhCCL2 and the respective anti-CCL2 antibody (with the two different Fcparts: SG1=IgG1 wild type with intact Fc gamma receptor binding andSG105=IgG1 with no Fc gamma receptor binding) into FcRn transgenic mice.The antibody profiles were analyzed by non-compartmental analysis usingPhoenix 64 (Pharsight/Certara). The AUCinf was estimated by linear-logtrapezoidal rule extrapolated to infinity. Clearance values are definedas Dose/AUCinf. This difference in clearance was also expressed as foldchange, which is calculated by dividing the hCCL2 clearance ofantibodies with Fc gamma receptor binding (SG1) by the hCCL2 clearanceof antibodies with Fc gamma receptor binding silent (SG105) (Table 3below). The data in the Table 3 below indicates that the clearance ofhuman CCL2 by Fc gamma receptor binding antibodies (SG1=IgG1 wild typewith intact Fc gamma receptor binding) was similar to that by Fc gammareceptor binding silent antibodies (SG105, with no Fc gamma receptorbinding) for all the monoparatopic antibodies tested. This suggests thatimmune complex-mediated sweeping of CCL2 by the tested monoparatopicantibodies was not efficient.

TABLE 3 Clearance values of wild type CCL2 after administration ofpre-formed immune complex of anti-CCL2 monospecific antibody (20 mg/kg)and wild type human CCL2 (0.1 mg/kg) (either IgG1 wild type (SG1) orIgG1 Fc receptor silenced (SG105) Fold Change (wild type IgG1 (SG1) vsFc receptor Clearance silenced IgG1 (ml/day/kg) (SG105)) CNTO888-SG114.05 1.60 CNTO888-SG105 8.80 11K2-SG1 80.60 1.69 11K2-SG105 47.83ABN912-SG1 67.06 0.99 ABN912-SG105 67.84 1A4-SG1 38.93 1.66 1A4-SG10523.39 1A5-SG1 20.08 0.95 1A5-SG105 21.14 1G9-SG1 14.02 0.87 1G9-SG10516.07 2F6-SG1 21.49 1.03 2F6-SG105 20.94Measurement of Total Human CCL2 Concentration in Serum byElectrochemiluminescence (ECL)

The concentration of total human CCL2 in mouse serum was measured byECL. 3 ug/mL of anti-CCL2 antibody (F7 (Biolegend) or clone MAB679 (R&DSystems)) was immobilized onto a MULTI-ARRAY 96-well plate (Meso ScaleDiscovery) overnight before incubating in blocking buffer for 2 hours at30° C. Anti-CCL2 MAB679 was used as capture antibody for samplescontaining humanized 11K2, 1A4 or 1A5 antibodies. Anti-CCL2 clone 5D3-F7was used for samples containing ABN912, CNTO888, 1G9, 2F6H antibodies.Human CCL2 calibration curve samples, quality control samples and mouseserum samples were prepared by diluting in dilution buffer andincubating with excess drug for 30 minutes at 37° C. After that, thesamples were added onto anti-CCL2-immobilized plate, and allowed to bindfor 1 hour at 30° C. before washing. Next, SULFO TAG NHS-ester (MesoScale Discovery) labelled anti-human Fc (clone: JDC-10, SouthernBiotech)was added and the plate was incubated for 1 hour at 30° C. beforewashing. Read Buffer T (×4) (Meso Scale Discovery) was immediately addedto the plate and signal was detected by SECTOR Imager 2400 (Meso ScaleDiscovery). The human CCL2 concentration was calculated based on theresponse of the calibration curve using the analytical software SOFTmaxPRO (Molecular Devices).

Measurement of Anti-CCL2 Antibody Concentration in Serum byEnzyme-Linked Immunosorbent Assay (ELISA)

The concentration of anti-CCL2 antibody in mouse serum was measured byELISA. Anti-human IgG kappa-chain (Antibody Solutions) was dispensedonto a Nunc MaxiSorp plate (Thermofisher) and allowed to stand overnightat 4 degrees C. to prepare anti-human IgG-immobilized plates.Calibration curve and samples were prepared with 1% pooled mouse serum.Then, the samples were dispensed onto the anti-human IgG-immobilizedplates, and allowed to stand for 1 hour at 30 degrees C. Subsequently,goat anti-human IgG (gamma-chain specific) with HRP conjugate (SouthernBiotech) was added to react for 1 hour at 30 degrees C. Chromogenicreaction was carried out using TMB substrate (Life Technologies) as asubstrate. After stopping the reaction with 1 N sulfuric acid (Wako),the absorbance at 450 nm was measured by a microplate reader. Theconcentration in mouse plasma was calculated from the absorbance of thecalibration curve using the analytical software SOFTmax PRO (MolecularDevices).

Evaluation of Endogenous Mouse CCL2 Immune Complex Sweeping withMonoparatopic Antibody in Mice

In addition to the results above (which suggests that immunecomplex-mediated sweeping of CCL2 by the tested monoparatopic antibodieswas not efficient) a further evaluation was conducted.

To evaluate the ability of monoparatopic antibodies to form and clearimmune complex with endogenous mouse CCL2, mouse cross-reactive 11K2anti-CCL2 monoparatopic antibodies was administered to mice. Humanized11K2H2-SG1 (IgG1 wild type=Fc gamma receptor binding) or humanized11K2-SG105 (Fc gamma receptor binding silent) antibodies wereintravenously administered at a single dose of 20 mg/kg at a single doseof 10 ml/kg into the caudal vein of Balb/c mice. Blood was collectedpre-administration, 5 minutes, 7 hours, 1 day, 2 days, 3 days and 7 daysafter administration. Serum was prepared by centrifuging the bloodimmediately at 14,000 rpm for 10 minutes in 4° C. The serum was storedat or below −80° C. until measurement.

FIG. 3 a and FIG. 3 b show the time course of serum total mouse CCL2concentration and antibody-time profile for humanized 11K2-SG1 and11K2-SG105 in mice.

As seen in FIG. 3 a and FIG. 3 b , the levels of accumulated mouse CCL2was not different between 11K2-SG105 (Fc gamma receptor binding silentFc) and 11K2-SG1 (IgG1 wild type=Fc gamma receptor binding Fc). Thisindicates that there was no or little Fc gamma receptor-mediatedclearance of endogenous mouse CCL2 by the injected antibodies. Asantigens in immune complexes are cleared more rapidly than uncomplexedantigens via multimeric engagement of Fc gamma receptors, this suggeststhat the 11K2 antibody was not able to form immune-complexes withendogenous mouse CCL2.

Measurement of Mouse CCL2 Concentration in Mouse Serum by Enzyme-LinkedImmunosorbent Assay (ELISA)

The concentration of mouse CCL2 in mouse serum was measured by adaptingthe reagents from a commercially available mouse CCL2 ELISA kit (R&DSystems). The manufacturer's protocol was followed except forpreparation of calibration curve samples. Purified recombinant mouseCCL2 was substituted as the standard instead of the manufacturer'sprotein. For samples taken after antibody was injected, calibrationcurve samples and samples were prepared with 2.5% mouse serum injectedantibody spiked in at a concentration of 40 microgram/ml, and incubatedfor 30 minutes at 37 degrees C. Subsequently, the samples were dispensedonto the anti-human CCL2-immobilized plates, and incubated at 30 degreesC. for 2 hours. Detection by adding mouse MCP-1 conjugate and incubatingfor 30 degrees C. for 2 hours, followed by substrate and stop solution.

For samples taken before antibody was injected, Mouse MCP-1Ultra-Sensitive Kit (Meso Scale Discovery) was used according to themanufacturer's instructions. No antibody was spiked into the samplebefore addition to the plate.

Measurement of Anti-CCL2 Antibody Concentration in Serum byEnzyme-Linked Immunosorbent Assay (ELISA)

The concentration of anti-CCL2 antibody in mouse serum was measured byELISA. Anti-human IgG kappa-chain (Antibody Solutions) was dispensedonto a Nunc MaxiSorp plate (Thermofisher) and allowed to stand overnightat 4 degrees C. to prepare anti-human IgG-immobilized plates.Calibration curve and samples were prepared with 1% pooled mouse serum.Then, the samples were dispensed onto the anti-human IgG-immobilizedplates, and allowed to stand for 1 hour at room temperature.Subsequently, mouse anti-human IgG HRP (clone JDC-10, Southern Biotech)was added to react for 30 minutes at room temperature. Chromogenicreaction was carried out using ABTS substrate (KPL) as a substrate andthe absorbance at 405 nm was measured by a microplate reader. Theconcentration in mouse plasma was calculated from the absorbance of thecalibration curve using the analytical software SOFTmax PRO (MolecularDevices)

Conclusion of the Different Mouse PK Studies with Monospecific(Monoparatopic) Anti-CCL2 Antibodies

To summarize the results of the mouse PK studies, none of themonoparatopic antibodies tested did show efficient clearance of CCL2from the circulation. These data suggest that monoparatopic antibodiesare not able to form immune complexes with CCL2 to efficiently clear itfrom the circulation.

In contrast, as described below, bispecific anti-CCL2 antibodies withtwo different antigen binding moieties/sites (biparatopic anti-CCL2antibodies) were able to efficiently form immune complexes with CCL2 andclear it from the circulation.

Example B-1

Bispecific (Biparatopic) Anti-CCL2 Antibodies

Several Bispecific Anti-CCL2 Antibodies with Two Different AntigenBindings Moieties (Paratopes) Binding to Two Different Specific Epitopeson Human CCL2 were Generated

Introduction

To test whether single binding or cross-linking of the antigen has asignificant impact on the in vivo CCL2 clearance, we generatedbispecific anti-CCL2 antibodies with 2 different antigen-bindingmoieties/sites that bind to 2 different epitopes on CCL2 using thebispecific CrossMab Technology (see e.g., WO 2009/080252, WO2015/150447), WO 2009/080253, WO 2009/080251, WO 2016/016299, Schaeferet al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016)1010-20) (bispecific (=biparatopic) CrossMabs). These molecules werefirst characterized in vitro for their biochemical and functionalproperties but they also served as tools for an in vivo CCL2 clearanceevaluation in a mouse co-injection study. To evaluate the clearancepotential based on Fcgamma Receptor (FcgR) binding mediated sweeping(e.g. in Igawa et al, Immunological Reviews 270 (2016) 132-151,WO2012/122011 and WO2016/098357 and WO2013/081143) we generated allCrossmabs as wild type huIgG1 which bind to FcgR and with modified humanIgG1 constant chain which have reduced/abolished binding to FcgReffector silent molecules (e.g. IgG1 with mutations L234A, L235A, P329G(Kabat EU numbering).

Identification of Suitable Anti-CCL2 Antibody Pairs—Selection ofBiparatopic Antibody Arms by Sandwich ELISA.

Sandwich ELISA was performed to identify antibody pairs that do notcompete for binding to human CCL2. 384-well MAXISORP (NUNC) plates werecoated with 1 μg/mL of the 7 indicated capture antibodies (Arm 1) andblocked with 2% BSA. Biotinylated (NHS-PEO₄-Biotin, Pierce) WT humanCCL2 (20 ng/mL) was incubated with excess amount of the same 7antibodies (Arm 2) at 1 μg/mL or block buffer for 1 hour at 37 degreesCelsius. After incubation, the mixtures were added to the blocked ELISAplate and incubated for 1 hour at room temperature. Detection of platebound CCL2 was performed using streptavidin HRP followed by TMB OneComponent substrate (Lifetech). Signal development was stopped by 1N HClacid (Wako). The O.D. of wells with no competing antibody was set as100% signal for each capture antibody. The O.D. of blank wells with noCCL2 added was set as 0% signal. Nine antibody pairs that did not showstrong competition for CCL2 binding in both directions were selected ascandidates for generation of bispecific Crossmab antibodies.

Antibody Arm 2 ABN CNTO 912 888 11K2 1A4 1A5 1G9 2F6 Antibody ABN912selected selected Arm 1 CNTO888 selected selected 11K2 selected selected1A4 selected 1A5 selected selected 1G9 2F6Generation and Characterization of Biparatopic Anti-CCL2 Antibodies andImmune ComplexesGeneration of Biparatopic Anti-CCL2 Antibodies in Bispecific CrossMabFormat Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene and Oligonucleotide Synthesis

Desired gene segments were prepared by chemical synthesis at GeneartGmbH (Regensburg, Germany). The synthesized gene fragments were clonedinto an E. coli plasmid for propagation/amplification. The DNA sequencesof subcloned gene fragments were verified by DNA sequencing.Alternatively, short synthetic DNA fragments were assembled by annealingchemically synthesized oligonucleotides or via PCR. The respectiveoligonucleotides were prepared by metabion GmbH (Planegg-Martinsried,Germany)

Description of the Basic/Standard Mammalian Expression Plasmid

For the expression of a desired gene/protein (e.g. antibody heavy chainor antibody light chain) a transcription unit comprising the followingfunctional elements is used:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   a gene/protein to be expressed (e.g. full length antibody heavy        chain or MHC class I molecule), and    -   the bovine growth hormone polyadenylation sequence (BGH pA).    -   Beside the expression unit/cassette including the desired gene        to be expressed the basic/standard mammalian expression plasmid        contains    -   an origin of replication from the vector pUC18 which allows        replication of this plasmid in E. coli, and    -   a beta-lactamase gene which confers ampicillin resistance in E.        coli.        Generation of Expression Plasmids for Recombinant Monoclonal        Antibodies

The recombinant monoclonal antibody genes encode the respectiveimmunoglobulin heavy and light chains.

The expression plasmids for the transient expression monoclonal antibodymolecules comprised besides the immunoglobulin heavy or light chainexpression cassette an origin of replication from the vector pUC18,which allows replication of this plasmid in E. coli, and abeta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of a respective antibody heavy or light chaincomprised the following functional elements:

-   -   the immediate early enhancer and promoter from the human        cytomegalovirus (P-CMV) including intron A,    -   a human heavy chain immunoglobulin 5′-untranslated region        (5′UTR),    -   a murine immunoglobulin heavy chain signal sequence,    -   the respective antibody heavy or light chain cDNA sequence and    -   the bovine growth hormone polyadenylation sequence (BGH pA).        Transient Expression and Analytical Characterization

The recombinant production was performed by transient transfection ofHEK293 cells (human embryonic kidney cell line 293-derived) cultivatedin F17 Medium (Invitrogen Corp.). For the production of monoclonalantibodies, cells were co-transfected with plasmids containing therespective immunoglobulin heavy and light chain. For transfection“293-Fectin” Transfection Reagent (Invitrogen) was used. Transfectionwas performed as specified in the manufacturer's instructions. Cellculture supernatants were harvested three to seven (3-7) days aftertransfection. Supernatants were stored at reduced temperature (e.g. −80°C.).

General information regarding the recombinant expression of humanimmunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al.,Biotechnol. Bioeng. 75 (2001) 197-203. To generate the followingbispecific antibodies, the CrossMab technology described in WO2016/016299 was used, in which VH/VL have been exchanged in one antibodyarm and the CH1/CL interface of the other antibody arm has been modifiedby charge modifications, in combination with the knobs-into-holestechnology in the CH3/CH3 interface to foster heterodimerization. Anexemplary sequence for all four antibody chains where this technologywas applied is given for CNTO888/11K2-WT IgG1 (see SEQ ID NO: 104 to SEQID NO:107)

List of Generated Bispecific (Biparatopic) Anti-CCL2 Crossmab Antibodieswith Wild Type IgG1 (WT IgG1) (Wild Type IgG1 Means withoutModifications/Mutations which Influence Fc Receptor Binding, HoweverHeterodimerization Technology Like Knobs into Holes is Included)

Second antigen binding site VH/VL Bispecific First antigen normalantibody binding site structure IgG1 Alias l Alias 2 VH/VL_crossed(non-crossed) ABN912//11K2- CCL2- P1AA3447 humanized ABN912 WT IgG1 004711K2 CNTO888//11K2- CCL2- P1AA3429 humanized CNTO888 WT IgG1 0048 11K211K2//1G9-WT CCL2- P1AA3461 humanized 1G9 IgG1 0049 11K2 11K2//2F6-WTCCL2- P1AA3392 humanized 2F6 IgG1 0056 11K2 CNTO888//1A5- CCL2- P1AA3419CNTO888 1A5 WT IgG1 0050 CNTO888//1G9- CCL2- P1AA3439 CNTO888 1G9 WTIgG1 0051 1A4//2F6-WT CCL2- P1AA3400 1A4 2F6 IgG1 0057 1A5//2F6-WT CCL2-P1AA3427 1A5 2F6 IgG1 0058 1A5//1G9-WT CCL2- P1AA3446 1A5 1G9 IgG1 0052List of Bispecific (Biparatopic) Anti-CCL2 Crossmab Antibodies with IgG1Including the Fc Gamma Receptor Silencing Mutations L234A, L235A, P329G(Kabat EU Numbering) (IgG1-PGLALA)

Second antigen binding site Bispecific VH/VL antibody First antigennormal IgG1 binding site structure PGLALA Alias 1 Alias 2 VH/VL_crossed(non-crossed) ABN912//11K2- CCL2- P1AA3411 humanized ABN912 PGLALA 004111K2 CNTO888//11K2- CCL2- P1AA3452 humanized CNTO888 PGLALA 0042 11K211K2//1G9- CCL2- P1AA3463 humanized 1G9 PGLALA 0043 11K2 11K2//2F6-CCL2- P1AA3450 humanized 2F6 PGLALA 0053 11K2 CNTO888//1A5- CCL2-P1AA3448 CNTO888 1A5 PGLALA 0044 CNTO888//1G9- CCL2- P1AA3455 CNTO8881G9 PGLALA 0045 1A4//2F6- CCL2- P1AA3459 1A4 2F6 PGLALA 0054 1A5//2F6-CCL2- P1AA3402 1A5 2F6 PGLALA 0055 1A5//1G9- CCL2- P1AA3444 1A5 1G9PGLALA 0046Purification of the Biparatopic Anti-CCL2 Antibodies

Biparatopic anti-CCL2 antibodies containing cell culture supernatantswere filtered and purified by up to three chromatographic steps.Depending on the purity of the capture step eluate an ion exchangechromatography step was optionally implemented between capture andpolishing step.

Biparatopic anti-CCL2 antibodies were purified from cell culturesupernatants by affinity chromatography using MabSelectSure-Sepharose™(GE Healthcare, Sweden), POROS 50 HS (Thermofisher Scientific) andSuperdex 200 size exclusion (GE Healthcare, Sweden) chromatography.Briefly, sterile filtered cell culture supernatants were captured on aMabSelect SuRe resin equilibrated with PBS buffer (10 mM Na2HPO4, 1 mMKH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibrationbuffer and eluted with 25 mM sodium citrate at pH 3.0. The elutedprotein fractions were pooled and neutralized with 2M Tris, pH 9.0. Ionexchange chromatography as optional second purification step wasperformed with POROS 50 HS (Thermofisher Scientific), equilibration andwash with 20 mM histidine pH 5.6 and load of diluted capture step eluatea gradient chromatography was done with 20 mM histidine, 0.5M NaCl at pH5.6. ion exchange chromatography fractions were analyzed by CE-SDSLabChip GX II (PerkinElmer) and Crossmab containing fractions werepooled.

Size exclusion chromatography on Superdex 200 (GE Healthcare) was usedas second or third purification step. The size exclusion chromatographywas performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Sizeexclusion chromatography fractions were analyzed by CE-SDS LabChip GX II(PerkinElmer) and Crossmab containing fractions were pooled and storedat −80° C.

In case of a satisfying product quality after the POROS 50 HS(ThermoFisher Scientific) size exclusion chromatography on Superdex 200(GE Healthcare) replaced by desalting chromatography on HiPrep 26/10Desalting (GE Healthcare) in 20 mM histidine buffer, 0.14 M NaCl, pH6.0.

The protein concentration of antibody preparations was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

Purity and integrity of the antibodies were analyzed by CE-SDS using aLabChip GX II (PerkinElmer) with Protein Express Chip and HT ProteinExpress Reagents Kit. Aggregate content of antibody preparations wasdetermined by high-performance SEC using a Biosuite High Resolution SEC,250 Å, 5 μm analytical size-exclusion column (Waters GmbH) using 200 mMK2HPO4/KH2PO4, 250 mM KCl, pH 7.0 as running buffer. Average puritieswere between 94-100% as analyzed by CE-SDS and monomer content >95%(SEC).

Functional Characterization of the Bispecific (Biparatopic) Anti-CCL2Antibodies Affinity Measurement (Binding)

Around 1200 resonance units (RU) of the capturing system (20 μg/ml goatanti human IgG Fc; Order Code: 109-005-098; Jackson Immuno Research)were coupled on a C1 chip (GE Healthcare BR-1005-35) at pH 5.0 by usingan amine coupling kit supplied by GE Healthcare. The sample and systembuffer was PBS-T (10 mM phosphate buffered saline including 0.05%Tween20) pH 7.4. The flow cell was set to 25° C.—and the sample blockset to 12° C.—and primed with running buffer twice. The bispecificantibody was captured by injecting a 2 μg/ml solution for 60 sec at aflow rate of 10 μl/min. Association was measured by injection of humanCCL2 (wt) in various concentrations in solution for 150 sec at a flowrate of 30 μl/min starting with 30 nM in 1:10 dilutions. Thedissociation phase was monitored for up to 1200 sec and triggered byswitching from the sample solution to running buffer. The surface wasregenerated by 60 sec washing with a 0.85% H₃PO₄ solution at a flow rateof 10 μl/min. Bulk refractive index differences were corrected bysubtracting the response obtained from a goat anti human IgG Fc surface.Blank injections are also subtracted (=double referencing). Forcalculation of kinetic parameters, the Langmuir 1:1 model was used.

Mono and Ligand bispecific anti- Level Rmax CCL2 antibodies Alias t 1/2*(min) (RU) (RU) Humanized 11K2 CCL2-0002 no dissociation 293.5 39.3(=11K2) ABN912 CCL2-0003 27 307.0 33.6 CNTO888 CCL2-0004 no dissociation272.8 31.9 ABN912//11K2- CCL2-0041 no dissociation 212.2 21.1 PGLALACNTO888//11K2- CCL2-0042 no dissociation 211.8 22.0 PGLALA 1IK2//1G9-CCL2-0043 no dissociation 204.9 21.2 PGLALA CNTO888//1A5- CCL2-0044 nodissociation 207.3 20.0 PGLALA CNTO888//1G9- CCL2-0045 no dissociation213.0 19.0 PGLALA 1A5//1G9- CCL2-0046 no dissociation 203.1 20.0 PGLALAABN912//11K2- CCL2-0047 no dissociation 209.3 20.2 WT IgG1CNTO888//11K2- CCL2-0048 no dissociation 226.1 22.9 WT IgG1 11K2//1G9-WTCCL2-0049 no dissociation 206.1 21.1 IgG1 CNTO888//1A5- CCL2-0050 nodissociation 214.4 20.3 WT IgG1 CNTO888//1G9- CCL2-0051 no dissociation215.8 20.7 WT IgG1 1A5//1G9-WT CCL2-0052 no dissociation 211.7 21.0 IgG111K2//2F6- CCL2-0053 no dissociation 201.9 19.8 PGLALA 1A4//2F6-CCL2-0054 no dissociation 194.7 18.8 PGLALA 1A5//2F6- CCL2-0055 nodissociation 203.5 20.1 PGLALA 11K2//2F6-WT CCL2-0056 no dissociation205.3 20.2 IgG1 1A4//2F6-WT CCL2-0057 no dissociation 199.0 19.5 IgG11A5//2F6-WT CCL2-0058 no dissociation 202.8 19.8 IgG1Natural Immune Complex Formation in the Presence of Wild Type Antigen.

All protein samples (bispecific anti-CCL2 CrossMab antibodies andantigens) were re-buffered in 1×PBS, pH 7.4, using dialysis orcentrifugal ultrafiltration devices.

A dilution series of the CrossMab samples from 2.0 to 0.1 mg/mL wasprepared. Likewise, antigen solutions in PBS were prepared withconcentrations ranging from 0.012 to 0.23 mg/mL. Concentrations werechosen to allow mixing of equivalent volumes to achieve a constant molarratio of 1:1 (antibody:CCL2 complex). The following antigen was used inthis study: wild type CCL2.

Equivalent volumes of the pre-diluted CrossMab and CCL2 preparation weremixed and incubated at 37° C. for 1 hour before samples were applied ona Superose6 (GE Healthcare #2039) column, pre-equilibrated with PBS andeluted at a flow rate of 0.5 mL/min. A total of 100 μg or the maximalpossible volume of 250 μL was applied and the antibodies and antigensalone were used as a control.

SEC-MALLS data were recorded with an OptiLab rEX refractive indexdetector and with a miniDAWN Treos MALLS detector (both from Wyattinc.). SEC-MALLS signals were processed using the Astra V5 software(Wyatt).

Interaction with Bispecific anti-CCL2 CCL2wt antibody Alias (low conc.)ABN912//11K2-WT IgG1 CCL2-0047 + CNTO888//11K2-WT IgG1 CCL2-0048 +++11K2//1G9-WT IgG1 CCL2-0049 ++ CNTO888//1A5-WT IgG1 CCL2-0050 +CNTO888//1G9-WT IgG1 CCL2-0051 + 1A5//1G9-WT IgG1 CCL2-0052 ++11K2//2F6-WT IgG1 CCL2-0056 ++ 1A4//2F6-WT IgG1 CCL2-0057 + 1A5//2F6-WTIgG1 CCL2-0058 + Legend +++ large quantity of multi-/oligomers ++ mediumquantity of multi-/oligomers + low quantity of multi-/oligomers 0 onlydimers or lessCCR2 Reporter Assay to Study the Neutralizing Characteristics ofAnti-CCL2 Antibodies

Tango™ CCR2-bla U2OS cells were purchased from Invitrogen, Germany, tostudy the impact of CCL2 neutralizing antibody constructs. Thosereporter cells contain the human Chemokine (C—C Motif) Receptor 2 (CCR2)linked to a TEV protease site and a Gal4-VP16 transcription factorstably integrated into the Tango™ GPCR-bla U2OS parental cell line. Thisparental cell line stably expresses a beta-arrestin/TEV protease fusionprotein and the beta-lactamase (bla) reporter gene under the control ofa UAS response element. Adding the natural ligands MCP1=CCL2 resulted inan indication of the activity of the reporter gene, which can bemeasured by the cleavage of a FRET-enabled substrate.

Principally, assay and cell handling procedures were according theproviders manual. In brief, CCR2-U2OS cells were seeded at a density of2×10⁴ cells/well (96er black clear bottom plate, cat. no. #655090,Greiner Bio-one) in 50 μl assay medium (Freestyle 293 Expression Medium,cat. no. #12338-018, Invitrogen). In parallel, serial dilutions ofdifferent test antibodies as well as the CCL2-antigen solution wereprepared at c=4× final concentration. Then, CCL2-antigen/antibodymixtures were prepared and pre-incubated for two to three hours (hrs) atRT. 50 μl of indicated CCL2/antibody solutions were transferred to theCCR2 expressing U2OS cells and incubated for 18 hrs in a humidifiedincubator at 37° C. and 5% CO₂. As control only assay medium was used.

On the next day, the CCF4 substrate (cat. no. #K1089, Invitrogen) wasprepared with ß-lactamase loading solution (cat. no. #K1085, Invitrogen)and 20 μl/well thereof were added to the cells. The substrate solutionwas incubated for two hrs at RT in the dark.

Finally, the fluorescence wavelengths were determined with a Spectra Max(M4) reader (Molecular devices) at the following wavelengths (Ex/Em=409nm/460 nm=blue*; Ex/Em=409 nm/530 nm=green**) and the ratio ofblue/green fluorescence after subtracting assay medium control wascalculated according to the following equation:ratio=(sample-blue*−control-blue*)/(sample-green**−control-green**).

After the pH-engineering we characterized the final LO candidates(CKLO1-4) for their ability to inhibit CCL2-induced CCR2 signaling. Inthis case the neutralizing property was assessed by only the monomericvariant of the rec. CCL2 protein, which was used at a finalconcentration of approx. 15 ng/ml.

Bispecific IC50 [ng/ml] Antibody Alias Exp 1 Exp 2 Exp3 ABN912//11K2-CCL2-0041 80.2 PGLALA CNTO888//11K2- CCL2-0042 84 PGLALA 11K2//1G9-CCL2-0043 57 PGLALA CNTO888//1A5- CCL2-0044 77.7 88 PGLALA CNTO888//1G9-CCL2-0045 86.4 81.3 PGLALA 1A5//1G9- CCL2-0046 75.8 PGLALA ABN912//11K2-CCL2-0047 84.4 WT IgG1 CNTO888//11K2- CCL2-0048 92.9 WT IgG111K2//1G9-WT CCL2-0049 82.2 IgG1 CNTO888//1A5- CCL2-0050 89.2 WT IgG1CNTO888//1G9- CCL2-0051 89.6 52.7/67.9 WT IgG1 1A5//1G9-WT CCL2-005286.2 IgG1 11K2//2F6- CCL2-0053 26.3 49.6/70   PGLALA 1A4//2F6- CCL2-005478.2 PGLALA 1A5//2F6- CCL2-0055 48.7 PGLALA 11K2//2F6-WT CCL2-0056 93.9IgG1 1A4//2F6-WT CCL2-0057 58.2 IgG1 1A5//2F6-WT CCL2-0058 93 IgG1Monospecific CCL2-0002 76.1 79.1 56.9/58   controlEvaluation of Human CCL2 Immune Complex Sweeping with BiparatopicAntibody in Mice

To evaluate the ability of biparatopic antibodies to form immune complexwith wild type human CCL2, pre-formed immune complexes consisting ofanti-CCL2 biparatopic antibody (20 mg/kg) and wild type human CCL2 (0.1mg/kg) were administered at a single dose of 10 ml/kg into the caudalvein of Balb/c mice. Blood was collected 5 minutes, 7 hours, 1 day, 3days and 7 days after administration. Serum was prepared by centrifugingthe blood immediately at 14,000 rpm for 10 minutes in 4° C. The serumwas stored at or below −80° C. until measurement. The biparatopicantibodies tested are listed in the Table 4 below. Antibodies with WTIgG1 Fc have Fc gamma receptor binding similar to wild-type whileantibodies with PGLALA Fc are Fc gamma receptor binding silent. Resultsare shown in FIG. 4 a -FIG. 4 i.

The effect of immune complex sweeping of each anti-CCL2 biparatopicantibody on hCCL2 clearance in vivo were assessed by comparing anti-CCL2antibody with Fc gamma receptor binding (solid line) and anti-CCL2antibody with Fc gamma receptor binding silent (PGLALA, dotted line), asshown in FIG. 4 a -FIG. 4 i . The antibody profiles were analyzed bynon-compartmental analysis using Phoenix 64 (Pharsight/Certara). TheAUCinf was estimated by linear-log trapezoidal rule extrapolated toinfinity. Clearance values are defined as Dose/AUCinf. This differencein clearance was also expressed as fold change, which is calculated bydividing the hCCL2 clearance of antibodies with Fc gamma receptorbinding (SG1) by the hCCL2 clearance of antibodies with Fc gammareceptor binding silent (PGLALA) (Table 4 below). The data in the Table4 below indicates that the clearance of human CCL2 by Fc gamma receptor(FcgR) binding antibodies (WT IgG1) was superior to that by Fc gammareceptor binding silent antibodies (PGLALA, with an IgG1 Fc domaincomprising mutation L234A, L235A, P329G mutations (Kabat EU numbering))for all the biparatopic antibodies tested. This suggests that immunecomplex-mediated sweeping of CCL2 achieved by the tested biparatopicantibodies was more efficient. Moreover, several biparatopic antibodiesshowed large fold change in clearance values, for example,CNTO/humanized 11K2 (CNTO/11K2).

Fold change is calculated by dividing the hCCL2 clearance of antibodieswith WT FcgammaR (FcgR) binding with hCCL2 clearance of antibodies withPGLALA. As shown in FIG. 4 a -FIG. 4 i and the Table 4 below, CNTO/11k2shows the largest fold change of 21.5 between the antibody with IgG1wild type (WT) which has FcgR binding and antibody which is FcgR bindingsilent (PGLALA). This suggests that immune complex-mediated sweeping byCNTO/11k2-WT IgG1 is the most efficient among all variants.

TABLE 4 Clearance values of wild type CCL2 after administration ofpre-formed immune complex of anti-CCL2 biparatopic antibody (20 mg/kg)and wild type human CCL2 (0.1 mg/kg) Clearance Fold Antibodies Alias(ml/day/kg) change 11K2//1G9-WT IgG1 CCL2-0049 101.761 6.111K2//1G9-PGLALA CCL2-0043 16.713 CNTO888//11K2-WT IgG1 CCL2-0048244.705 21.5 CNTO888//11K2-PGLALA CCL2-0042 11.374 CNTO888//1G9-WT IgG1CCL2-0051 31.085 7.8 CNTO888//1G9-PGLALA CCL2-0045 3.999 CNTO888//1A5-WTIgG1 CCL2-0050 7.098 3.8 CNTO888//1A5-PGLALA CCL2-0044 1.892 1A5//1G9-WTIgG1 CCL2-0052 194.481 11.4 1A5//1G9-PGLALA CCL2-0046 17.07311K2//2F6-WT IgG1 CCL2-0056 14.442 4.9 11K2//2F6-PGLALA CCL2-0053 2.964ABN912//11K2-WT IgG1 CCL2-0047 20.935 4.2 ABN912//11K2-PGLALA CCL2-00414.988 1A4//2F6-WT IgG1 CCL2-0057 19.606 3.3 1A4//2F6-PGLALA CCL2-00545.933 1A5//2F6-WT IgG1 CCL2-0058 10.254 2.7 1A5//2F6-PGLALA CCL2-00553.825Measurement of Total Human CCL2 Concentration in Serum byElectrochemiluminescence (ECL)

The concentration of total human CCL2 in mouse serum was measured byECL. 3 ug/mL of anti-CCL2 antibody 2F2-SG1 was immobilized onto aMULTI-ARRAY 96-well plate (Meso Scale Discovery) overnight beforeincubating in blocking buffer for 2 hours at 30° C. Human CCL2calibration curve samples, quality control samples and diluted mouseserum samples were incubated with denaturing buffer consisting of either9% SDS for 30 minutes at 37° C., or pH2.0-2.5 Glycine HCl buffer for 10minutes at 37° C. The purpose of the denaturing buffer is to dissociatehuman CCL2 from the biparatopic antibody. After that, the samples werediluted 10-fold and added onto anti-CCL2-immobilized plate, and allowedto bind for 1 hour at 30° C. before washing. Next, SULFO TAG labelledMCP-1 antibody was added and the plate was incubated for 1 hour at 30°C. before washing. Read Buffer T (×4) (Meso Scale Discovery) wasimmediately added to the plate and signal was detected by SECTOR Imager2400 (Meso Scale Discovery). The human CCL2 concentration was calculatedbased on the response of the calibration curve using the analyticalsoftware SOFTmax PRO (Molecular Devices).

Measurement of Anti-CCL2 Antibody Concentration in Serum byEnzyme-Linked Immunosorbent Assay (ELISA)

The concentration of anti-CCL2 antibody in mouse serum was measured byELISA. Anti-human IgG kappa-chain (Antibody Solutions) was dispensedonto a Nunc MaxiSorp plate (Thermofisher) and allowed to stand overnightat 4 degrees C. to prepare anti-human IgG-immobilized plates.Calibration curve and samples were prepared with 1% pooled mouse serum.Then, the samples were dispensed onto the anti-human IgG-immobilizedplates, and allowed to stand for 1 hour at 30 degrees C. Subsequently,goat anti-human IgG (gamma-chain specific) with HRP conjugate (SouthernBiotech) was added to react for 1 hour at 30 degrees C. Chromogenicreaction was carried out using ABTS substrate (KPL) as a substrate andabsorbance at 450 nm was measured by a microplate reader. Theconcentration in mouse plasma was calculated from the absorbance of thecalibration curve using the analytical software SOFTmax PRO (MolecularDevices).

Summary of the Studies

To summarize the mouse PK study data, sweeping of human CCL2 by thetested biparatopic antibodies was more efficient compared tomonoparatopic antibodies at the same dose. With the monoparatopicantibodies, there were minimal difference in antigen clearance betweenantibodies with WT FcgR binding and FcgR binding silent (Table 3). Incontrast, large difference in antigen clearance between biparatopicantibodies with WT FcgR binding and FcgR binding silent were obtained(Table 4), suggesting that the tested biparatopic antibodies could sweephuman CCL2 efficiently. The combination of CNTO888/11K2 was chosen forfurther antibody engineering as it demonstrated the largest fold chancein clearance.

Example B-2 Anti-CCL2 Antibodies with Modified Variable Domains and CDRs(Ion Dependent/pH Dependent Binding)

Modification Leading to Ion Dependent/pH Dependent Binding

To generate pH-dependent anti-CCL2 antibodies, histidine scanningmutagenesis was conducted for all CDRs of mAbs CNTO888 and humanized11K2. Each amino acid in the CDRs was individually mutated to histidineusing In-Fusion HD Cloning Kit (Clontech Inc. or Takara Bio company)according to the manufacturer's instructions. After confirming throughsequencing that each variant was mutated correctly, variants weretransiently expressed and purified by the following method: Recombinantantibodies were expressed transiently using Freestyle FS293-F cells and293Fectin (Life technologies), according to the manufacturer'sinstructions. Recombinant antibodies were purified with protein A (GEHealthcare) and eluted in D-PBS or His buffer (20 mM Histidine, 150 mMNaCl, pH6.0). Size exclusion chromatography was further conducted toremove high molecular weight and/or low molecular weight component, ifnecessary. All histidine-substituted variants were evaluated by amodified BIACORE® assay as compared to that described above. Briefly, anadditional dissociation phase at pH5.8 was integrated into the BIACORE®assay immediately after the dissociation phase at pH7.4. This is toassess the pH-dependent dissociation between antibody (Ab) and antigen(Ag) from the complexes formed at pH7.4 as opposed to the correspondingdissociation at pH5.8. The dissociation rate at pH5.8 buffer wasdetermined by processing and fitting data using the Scrubber 2.0(BioLogic Software) curve fitting software.

Single histidine substitutions which resulted in reduction in bindingresponse at pH5.8 compared to the pH7.4 dissociation phase were selectedand combined. To identify mutations which improve affinity at pH7.4,more than 500 variants were generated for heavy and light chainrespectively, using at least one variant generated during the histidinesubstitution step. These variants had each amino acid in the CDRssubstituted with 18 other amino acids, excluding the original amino acidand Cysteine. The binding ability of variants to human CCL2 was assessedat 37 degrees C. under pH7.4 using BIACORE® 4000 instrument (GEHealthcare). As before, an additional dissociation phase at pH5.8 wasintegrated into the BIACORE® assay immediately after the dissociationphase at pH7.4. The dissociation rate at pH5.8 buffer was determined byprocessing and fitting data using the Scrubber 2.0 (BioLogic Software)curve fitting software.

Variants which improved affinity at pH7.4 and improved pH dependencywere selected, and these mutations were combined. This is exemplified infour 11K2 variants and four CNTO888 variants in the following Table.

TABLE Four modified 11K2 and four CNTO888 variants engineered forpH-dependent binding Modified 11K2 variants Modified CNTO888 variants(VH/VL) (VH/VL) 11K2H1503/11K2L1338 CNTO888H0625/CNTO888L061611K2H1510/11K2L1338 CNTO888H0634/CNTO888L0616 11K2H1503/11K2L1201CNTO888H0635/CNTO888L0616 11K2H1514/11K2L1338 CNTO888H0695/CNTO888L0616

To evaluate the combination effect of modified 11K2 and CNTO888variants, each 11K2 variant was combined with the four modified CNTO888variants, and expressed as a biparatopic CCL2 antibody in CrossMabformat. This is exemplified in the following Table, where the 4×4combination results in the generation of 16 biparatopic antibodies,designated as CKLO81 to CKLO16.

TABLE Combination of four modified 11K2 and four modified CNTO888variants to generate 16 bispecific (biparatopic) antibodies. Bispecificanti-CCL2 antibody Based on variable domains of CKLO0111K2H1503/11K2L1338//CNTO888H0695/CNTO888L0616 CKLO0211K2H1510/11K2L1338//CNTO888H0695/CNTO888L0616 CKLO0311K2H1503/11K2L1201//CNTO888H0695/CNTO888L0616 CKLO0411K2H1503/11K2L1201//CNTO888H0625/CNTO888L0616 CKLO0511K2H1503/11K2L1338//CNTO888H0634/CNTO888L0616 CKLO0611K2H1503/11K2L1201//CNTO888H0634/CNTO888L0616 CKLO0711K2H1514/11K2L1338//CNTO888H0634/CNTO888L0616 CKLO0811K2H1510/11K2L1338//CNTO888H0634/CNTO888L0616 CKLO0911K2H1503/11K2L1338//CNTO888H0625/CNTO888L0616 CKLO1011K2H1514/11K2L1338//CNTO888H0625/CNTO888L0616 CKLO1111K2H1510/11K2L1338//CNTO888H0625/CNTO888L0616 CKLO1211K2H1503/11K2L1338//CNTO888H0635/CNTO888L0616 CKLO1311K2H1503/11K2L1201//CNTO888H0635/CNTO888L0616 CKLO1411K2H1514/11K2L1338//CNTO888H0635/CNTO888L0616 CKLO1511K2H1510/11K2L1338//CNTO888H0635/CNTO888L0616 CKLO1611K2H1514/11K2L1338//CNTO888H0695/CNTO888L0616

To generate the bispecific antibodies, the CrossMab technology describedin WO 2016/016299 was used, in which VH/VL have been exchanged in oneantibody arm and the CH1/CL interface of the other antibody arm has beenmodified by charge modifications, in combination with the knobs-intoholes technology in the CH3/CH3 interface to foster heterodimerization.An exemplary sequence for all four antibody chains where this technologywas applied is given for CKLO2 IgG1 (see SEQ ID NO: 108 to SEQ IDNO:111). Depending on the heavy chain constant domain used (e.g. IgG1wild type (without Fc receptor binding silencing mutations), PGLALA,SG1095, SG1099, 1100—for SG1095, SG1099, 1100 see description below orsequence description) the suffixes IgG1, PGLALA, SG1095, SG1099, 1100are added

Functional Characterization of the Biparatopic Anti-CCL2 Antibodies withModified Variable Domains and CDRs (Ion Dependent/pH Dependent Binding)

Affinity Measurements (See Methods Above)

For all 16 generated bispecific anti-CCL2 antibodies as IgG1 wild typetheir pH dependent binding human CCL2 was determined.

FIG. 5 a shows Biacore® sensorgrams showing binding profile to monomericCCL2 at pH7.4 (black line) and pH5.8 (grey line) of the four modified11K2 and four CNTO888 variants, and the 16 Crossmabs after combination.

FIG. 5 b shows Biacore® sensorgrams showing binding profile of the fourmodified 11K2 and four CNTO888 variants, and the 16 Crossmabs aftercombination to monomeric CCL2, where an additional dissociation phase atpH5.8 was integrated into the BIACORE® assay immediately after thedissociation phase at pH7.4.

Cross-reactivity Binding to CCL8

pH dependency binding to recombinant monomeric human CCL2 andrecombinant monomeric human CCL8 were assessed at 37° C. using BiacoreT200 instrument (GE Healthcare). Anti-human Fc (GE Healthcare) wasimmobilized on each flow cells of a CM4 sensor chip using amine couplingkit (GE Healthcare) according to the recommended settings by themanufacturer. Antibodies and analytes were diluted into ACES pH 7.4 orpH 5.8 buffer (20 mM ACES, 150 mM NaCl, 1 mg/ml BSA, 0.05% Tween 20,0.005% NaN3). Antibodies were captured onto the anti-Fc sensor surfaces,then recombinant monomeric human CCL2 was injected over the flow cell at8 nM concentration. Association phase of analytes to antibodies wasmonitored for 120 s, followed by 180 s dissociation phase. Sensorsurface was regenerated each cycle with 3M MgCl₂. Binding sensorgram wasprocessed by TIBCO Spofire by normalization of binding response to thecapture level.

Assessment of pH-dependent dissociation for antibody/antigen complexesformed at pH 7.4 was checked by a modified Biacore assay. Briefly, anadditional dissociation phase at pH 5.8 was integrated into the Biacoreassay immediately after dissociation phase at pH 7.4. Binding sensorgramwas processed by TIBCO Spofire by normalization of binding response tothe capture level.

Expression and purification of recombinant human CCL8 P8A monomer: Thesequence for wild type human CCL8 was obtained from Genbank (NCBI:NP_005614.2). To make monomeric CCL8, proline at position 8 of themature CCL8 protein was mutated to alanine. Expi 293 cells (Lifetech)were transfected according to the manufacturer's instructions. CCL8 wildtype and P8A monomer protein were purified using the same method fromcell culture supernatants by cation exchange chromatography usingSP-Sepharose HP (GE Healthcare) and Superose 200 size exclusion (GEHealthcare) chromatography. Briefly, cell culture supernatants werediluted 2.5-fold with MilliQ water (Millipore), loaded on a Hi-TrapSP-HP column equilibrated with PBS, washed with equilibration buffer andeluted using a gradient of 0-2M NaCl. The eluted protein fractions werepooled and further purified by size exclusion chromatography using aHiLoad 16/600 Superose 200 (GE Healthcare) column equilibrated with 20mM histidine, 150 mM NaCl, pH 6.0. Fractions were analyzed by sizeexclusion chromatography and SDS-PAGE. Fractions containing CCL8 proteinwere pooled, concentrated and stored at −80° C.

Human CCL8 shares a high degree of homology with CCL2 and is able bindto CCR2 as well. As the 11K2 arm is able to bind CCL8 (see FIG. 1 , FIG.1A, FIG. 1B, and FIG. 1C), it was necessary to identify mutations toreduce this binding to avoid possible off-target effects of neutralizingCCL8. In addition, removal of CCL8 binding on the 11K2 arm is importantfor efficient formation of immune complex with CCL2. As the CNTO armdoes not bind CCL8, binding of CCL8 to the 11K2 arm will interfere withimmune complex formation with CCL2, which may reduce the clearance rateof CCL2 from plasma.

To identify mutations which reduce binding of 11K2 to human CCL8 andconfer selectivity to human CCL2, some CDR positions were substitutedlike e.g. D101E in the 111K2 VH of CKLO02 or W92R in the 11K2 VL ofCKLO03 to remove cross-reactivity to huCCL8. As shown in FIG. 6 , CCL8binding in the biparatopic Crossmab could be markedly reduced byengineering 11K2. The CKLO01 variant was not optimized to reduce CCL8binding, whereas CKLO04, CKLO03, and CKLO02, contain mutations to reduceCCL8 binding. All four Crossmabs have pH-dependent binding to CCL8.

Binding Affinity of Anti-CCL2 Antibodies to Recombinant CCL2 and CCL8 atpH 7.4 & pH 5.8

To determine the affinity and pH dependent binding of parentalCNTO888H/11K2H2, CKLO1, CKLO2 and CKLO3 to human CCL2 and CCL8 wasassessed at 37° C. using Biacore T200 instrument (GE Healthcare).Anti-human Fc (GE Healthcare) was immobilized on each flow cells of aCM4 sensor chip using amine coupling kit (GE Healthcare) according tothe recommended settings by the manufacturer. Antibodies and analyteswere diluted into ACES pH 7.4 or pH 5.8 buffer (20 mM ACES, 150 mM NaCl,1 mg/ml BSA, 0.05% Tween 20, 0.005% NaN3). Antibodies were captured ontothe anti-Fc sensor surfaces, then recombinant human CCL2 P8A variant(monomer) or CCL8 P8A variant (monomer) was injected over the flow cellat 1.25 nM to 20 nM prepared by two-fold serial dilution. Sensor surfacewas regenerated each cycle with 3M MgCl2. Binding affinity weredetermined by processing and fitting the data to 1:1 binding model usingBiacore T200 Evaluation software, version 2.0 (GE Healthcare). Thebinding affinity of anti-CCL2 antibodies to recombinant CCL2 and CCL8 atpH 7.4 & pH 5.8 are shown in the Table 5 below.

TABLE 5 Binding affinity of anti-CCL2 antibodies to recombinant CCL2 andCCL8 at pH 7.4 & pH 5.8 Bispecific anti- KD to human CCL2 KD to humanCCL8 CCL2 antibody pH 7.4 pH 5.8 pH 7.4 pH 5.8 CNTO8888/11k2 1.28E−112.35E−13 1.47E−09 1.75E−09 CKLO1 7.39E−12 9.32E−09 2.33E−08 n.d. CKLO24.32E−12 n.d. n.d. n.d. CKLO3 6.85E−12 2.17E−08 n.d. n.d. Note: n.d. KDcannot be determined due to low binding response.

The data in the Table 5 show that binding to CCL8 at pH7.4 was abolishedfor CKLO2 and CKLO3, while maintaining strong affinity at pH7.4 andpH-dependent binding for CCL2. The results show the differentmodifications introduced in the variable regions and CDRs of theparental bispecific antibody based on CNTO888 and 11K2 successfullygenerated affinity matured variants, CKLO1, CKLO2, CKLO3 with enhancedbinding affinity to CCL2 compared to parental Ab at pH 7.4. At the sametime CKLO1, CKLO2, CKLO3 showed strong pH dependent binding to CCL2. Amore than 1000-fold weaker KD of the binding affinity to CCL2 wasobserved at pH 5.8 compared to the KD value at pH 7.4

Clearance of Wild Type Human CCL2

To evaluate the ability of pH-dependent bispecific antibodies to enhancethe clearance of wild type human CCL2, pre-formed immune complexesconsisting of anti-CCL2 monoparatopic antibody (20 mg/kg) and wild typehuman CCL2 (0.1 mg/kg) were administered at a single dose of 10 ml/kginto the caudal vein of SCID mice. Blood was collected 5 minutes, 1hour, 4 hours, 7 hours, 1 day, and 7 days after administration. Theserum was stored at or below −80° C. until measurement. The Crossmabantibodies tested were parental CNTO/11K2, and four pH-engineeredvariants, CKLO01, CKLO02, CKLO03, and CKLO04. All antibodies had an IgG1wild type Fc part (without mutations silencing/abolishing Fc (gamma)receptor binding). Measurement of total human CCL2 and anti-CCL2antibody concentration in mouse serum was done as described above (under“Evaluation of human CCL2 immune complex sweeping with biparatopicantibody in mice” following Table 4).

Results are shown in FIG. 7 a : Serum concentration of hCCL2 over timeafter injection of pre-formed immune complex consisting of hCCL2 andbispecific anti-CCL2 antibodies (parental CNTO/11K2 and pH dependentvariants CKLO01, CKLO02, CKLO03 and CKLO04) into SCID mice. All fourpH-engineered variants showed rapid clearance of human CCL2. For CKL002,CKLO03, human CCL2 was below the detection limit by day 1. For theparental CNTO/11K2, rapid clearance of human CCL2 was initially observedtill day 1, but thereafter, clearance of human CCL2 was slow.

Example B-3 Anti-CCL2 Antibodies with Modified Variable Domains and CDRs(Ion Dependent/pH Dependent Binding) and Fc Mediated Sweeping

Modification of the Bispecific Anti-CCL2 Antibodies Via SweepingTechnology

The bispecific anti-CCL2 antibodies were modified using the sweepingtechnology to enable the bispecific anti-CCL2 antibodies to abrogatefree CCl2 over longer time periods to enable sustained a biologicaleffect like anti-cancer efficacy or anti-inflammatory efficacy in vivo.

The Sweeping concept is described e.g. in Igawa et al, ImmunologicalReviews 270 (2016) 132-151, WO2012/122011, WO2016/098357, andWO2013/081143 which are incorporated herein by reference.

pI Fc Mediated Sweeping

Having demonstrated the pH-engineered biparatopic antibodies canaccelerate CCL2 clearance in vivo, we next evaluated the ability ofantibodies with pI-increasing substitutions to enhance the clearance ofwild type human CCL2. Pre-formed immune complexes consisting ofanti-CCL2 monoparatopic antibody (20 mg/kg) and wild type human CCL2(0.1 mg/kg) were administered at a single dose of 10 ml/kg into thecaudal vein of SCID mice. Blood was collected 5 minutes, 30 minutes, 1hour, 2 hours, 4 hours, and 7 days after administration. The serum wasstored at or below −80° C. until measurement. The Crossmab antibodiestested were parental CKLO03 with IgG1, and CKLO03 with pI enhanced Fc,CKLO03-SG1099. Measurement of total human CCL2 and anti-CCL2 antibodyconcentration in mouse serum was done as described above (under“Evaluation of human CCL2 immune complex sweeping with biparatopicantibody in mice” following Table 4).

Results are shown in FIG. 7 b . Serum concentration of hCCL2 over timeafter injection of pre-formed immune complex consisting of hCCL2 andCKLO03 (with IgG1 wild type Fc) or CKLO03-SG1099, (CKLO03 with enhancedpI Fc) into SCID mice. CKLO03-SG1099, which contain Fc substitutionsQ311R/P343R (EU Kabat numb.) showed faster clearance/reduction of humanCCL2 compared to CKLO03 with IgG1. This demonstrates that pH-dependentbiparatopic antibody with pI-increasing mutations can accelerate theclearance of CCL2.

Generation of Biparatopic Anti-CCL2 Antibodies with FcgammaRIIb-EnhancedFc Variants and Further Fc Modifications

In this example, Fe engineering to enhance CCL2 clearance isillustrated.

It has been demonstrated in WO 2013/125667 that clearance of a solubleantigen can be enhanced by its administration of antigen-bindingmolecules (e.g. antibodies) comprising an Fc domain displaying anincreased affinity for FcgammaRIIb. Furthermore, Fc variants that canshow enhanced binding to human FcgammaRIIb have been illustrated in WO2012/115241 and WO 2014/030728. It has been also illustrated that theseFc variants can show selectively enhanced binding to human FcgammaRIIband decreased binding to other active Fc gamma Receptors (Fc gamma Rs).This selective enhancement of FcgammaRIIb binding can be favorable notonly for clearance of soluble antigen but also for decreasing the riskof undesired effector functions and immune response.

For development of an antibody drug, efficacy, pharmacokinetics, andsafety should be evaluated in non-human animals in which the drug ispharmacologically active. If it is active only in human, alternativeapproaches such as the use of a surrogate antibody must be considered(Int. J. Tox. 28: 230-253 (2009)). However, it would not be easy toprecisely predict the effects of the interaction between the Fc regionand Fc gamma Rs in human using a surrogate antibody, because theexpression patterns and/or functions of Fc gamma Rs in non-human animalsare not always the same as in human. It would be preferable that the Fcregions of antibody drugs should have cross-reactivity to non-humananimals, especially to cynomolgus monkey which has close expressionpatterns and functions of Fc gamma Rs to human, so that the resultsobtained in non-human animals could be extrapolated into human.

The following IgG1 constant domain/Fc variants of the bispecificanti-CCL2 antibodies were generated with mutations at positions of theFc part (EU Kabat numbering)

SG1095—derived from IgG1 including the mutations (Kabat EU numbering):

-   -   L235W/G236N/H268D/Q295L/A330K/K326T (suitable for increase        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)        SG1099—derived from IgG1 including mutations (Kabat EU        numbering):    -   Q311R/P343R (suitable for increasing pI for enhancing uptake of        antigen)        SG1100—derived from IgG1 including the mutations (Kabat EU        numbering):    -   Q311R/P343R (suitable for increasing pI for enhancing uptake of        antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody; and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)        GG01—derived from IgG1 including the mutations (Kabat EU        numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)        GG02—derived from IgG1 including mutations (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   M428L/N434A/Y436T (suitable for increasing affinity to FcRn for        longer plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)        GG03—derived from IgG1 (SG1-IgG1 allotype) including the        mutations (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   N434A (suitable for increasing affinity to FcRn for longer        plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)    -   GG04—derived from IgG1 (SG1-IgG1 allotype) including mutations        (Kabat EU numbering):    -   L234Y/P238D/T250V/V264I/T307P/A330K (suitable for increasing        affinity to human FcgRIIb and decreasing affinity to other human        FcgR);    -   Q311R/P343R (suitable for increasing isoelectric point (pI) for        enhancing uptake of antigen);    -   M428L/N434A/Y436T (suitable for increasing affinity to FcRn for        longer plasma half-life of antibody); and    -   Q438R/S440E (suitable for suppressing rheumatoid factor binding)        Functional Characterization of the Bispecific Anti-CCL2        Antibodies with Modified Variable Domains and CDRs (Ion        Dependent/pH Dependent Binding) and with or without Fc Mediated        Sweeping        SPR Binding of Fc Variants SG1095, GG01, GG02, GG03/04 of CKLO2

In an SPR assay at a Biacore 8K instrument, binding of monomeric humanand cyno CCL2 to the 4 different antibodies P1AD8325, P1AF8137,P1AF8139, and P1AF8140 at pH 7.4 and 5.8 was investigated.

In this set-up, CaptureSelect™ Human Fab-kappa (ThermoFisher Scientific)was immobilized on a CM3 sensor chip using the amine coupling method,the diverse antibodies were captured as ligands, and measurements wereperformed with 0, 10, 100 and 1000 nM monomeric human or cyno CCL2 as ananalyte at two different pH values.

CKLO2-SG1095, CKLO2-GG01, CKLO2-GG02, CKLO2-GG03/04 show almostidentical binding profiles to monomeric human and cyno CCL2 which bindat 10, 100 and 1000 nM and dissociate equally fast at pH 7.4, whereas nostable binding was observed at pH 5.8. Results are shown in the tablebelow.

TABLE Binding to monomeric human and cyno CCL2 of CKLO2-SG1095,CKLO2-GG01, CKLO2-GG02, CKLO2-GG03/04 show almost identical bindingprofiles to monomeric human and cyno CCL2 Rmax t 1/2 Rmax t 1/2 (RU) @(s) @ (RU) @ (s) @ Antibody Antigen pH 7.4 pH 7.4 pH 5.8 pH 5.8 CKLO2-Monomeric 15.9 6.18 29.8 3.29 GG01 human CCL2 CKLO2- Monomeric 16.8 4.0428.8 3.29 GG02 human CCL2 CKLO2- Monomeric 17.6 5.90 29.6 3.24 GG03/04human CCL2 CKLO2- Monomeric 19.0 5.49 35.3 3.51 SG1095 human CCL2 CKLO2-Monomeric 16.5 7.10 30.7 3.22 GG01, cyno CCL2 CKLO2- Monomeric 17.2 5.9729.5 3.14 GG02, cyno CCL2 CKLO2- Monomeric 17.9 6.48 30.4 3.12 GG03/04cyno CCL2 CKLO2- Monomeric 19.5 6.07 36.0 3.41 SG1095 cyno CCL2Chemotaxis AssayDescription of Method

THP-1 cells were cultured for 3 days up to 8×10E5 cells/ml. A total cellnumber of 5000 cells/well were seeded in the upper chamber of amicrotiter plate and let settle at 37° C. Recombinant huCCL2 waspipetted in the bottom chamber at a final concentration of 50 ng/ml inthe presence or not of anti-CCL2 antibodies (when the assay wasperformed to test surrogate antibodies, recombinant muCCL2 was usedinstead, at 100 ng/ml). Upper and bottom chambers were brought togetheravoiding the formation of air bubbles and the plate was then incubatedat 37° C. for 24 h. Migrated cells were quantified by the Cell Titer Glomethod according to manufacturer's recommendation, and luminescencemeasured with the Tecan Infinite 200 Reader.

Results are shown in FIG. 8 : Chemotaxis Assay: Bispecific anti-CCL2antibodies with identical CDRs and variable regions VH/VL, namelyCKLO2-IgG1 wild type and CKLO2-SG1095, but different Fc moieties, caninhibit the migration of THP-1 cells with identical potencies (IC₅₀=0.2μg/ml; FIG. 8 , left panel).

Similarly, CCL2-0048, the parent VH/VL-unmodified bispecific antibodyCNTO888/11k2 of CKLO2, which is non-pH dependent, also shows an IC₅₀ of0.2 μg/ml, since pH-dependency is critical for antigen sweeping, aphenomenon that does not take place in this assay.

The corresponding monoparatopic antibodies CNTO888 IgG1 and humanized11k2 IgG1 display IC₅₀ values of 0.3 and 0.7 μg/ml, respectively, whilethe huIgG1 isotype control shows no inhibition (FIG. 8 , right panel).

In additional analogous experiments the IC₅₀ values of CKLO2-GG01 (0.2μg/ml), CKLO2-GG02 (0.2 μg/ml), and GG03/GG04 (0.3 μg/ml), weredetermined.

In Vivo Biological Activity in a Genetically-Modified Mouse Model

Material and Methods

B16-huCCL2/CCL2-Null Model

The present model was generated with the aim of testing anti-human CCL2antibodies without the interference of mouse CCL2 in, otherwise, immunecompetent tumor-bearing mice. For this, the mouse tumor cell lineB16-F10 was chosen since it does not secrete mCCL2 and is known to growin vivo in mice of the C57/B16 strain, which is the genetic backgroundof the CCL2 knock-out mice.

To generate stable pools of B16F10 tumor cells expressing huCCL2 theywere transfected with plasmid DNA encoding for huCCL2 and a Hygromycin-Bselection cassette. Therefore, cells were seeded into 6-well plates with2.0E+05 cells/well in growth medium (DMEM+10% FCS+2 mM L-glutamine).After 24 h a transfection mix composed of 1 μg DNA per well andLipofectamine 2000 in Opti-MEM medium was added to the cells.Subsequently, the cells were put under selection with medium containingHygromycin-B (0.5 mg/ml). After 20 days of culture living single cellswere sorted based on FSC/SSC-scatter using a BD FACS Aria III. Twelvedays later, cell culture supernatants from single cell clones werescreened for expression of human CCL2 using the ELISA Ready-SET-Go fromebioscience (Cat #88-7399-86) in comparison to wild type B16F10 cells(data not shown).

The selected B16-F10_HOMSA_CCL2 tumor cell clones 1A5, 2A3 and 2B2 wereroutinely cultured in DMEM containing 10% FCS and 2 mM L-Glutamine (PANBiotech GmbH, Germany) at 37° C. in a water-saturated atmosphere at 5%CO2. Culture passage was performed with trypsin/EDTA 1×(PAN BiotechGmbH, Germany) splitting twice/week.

Female B6.129S4-Ccl2tm1Rol/J mice (Jackson Laboratories), age 7-10 weeksat arrival, were inoculated with the B16-F10_HOMSA_CCL2 tumor cellclones: on that day (study day 0), tumor cells were harvested fromculture flasks and transferred into culture medium, washed once andresuspended in PBS. Cell numbers were determined using a cell counterand analyzer system (Vi-CELL, Beckman Coulter). For s.c. injection celltiter was adjusted to 1×10E7 cells/ml and 100 μl were injectedsubcutaneously into the right flank of mice using a cooled

1.0 ml tuberculin syringe (Dispomed, Germany) and a small needle(0.45×12 mm). Cell inoculation was performed under general anesthesia byisoflurane (CP Pharma, Germany) in an inhalation unit for small animals.

Tumor growth was monitored daily and mice were sacrificed on study day15, when tumors reached about 1000 mm3 for B16-F10_HOMSA_CCL2 tumor cellclones 1A5 and 2A3 (at this time point 2B2 tumors were about 600 mm3 dueto a slower growth rate). At endpoint, blood samples were taken for CCL2measurement and tumors were explanted and analyzed by flow cytometry, asdescribed in above.

Mouse immune cells were found to infiltrate all tumors, confirming thenotion that human CCL2 is able to attract mouse CCR2+ cells.B16-F10_HOMSA_CCL2 tumor cell clone 1A5 displayed the highest CD45+total infiltrate with the highest relative mMDSC (monocyticmyeloid-derived suppressor cells) composition (FIG. 2 a -FIG. 2 g ).Clones 2A3 and 2B2 had lower frequencies of immune cells in the tumoreven though 2A3 cells led to similar levels of serum total CCL2 like 1A5cells, while the 2B2 clone showed a significantly lower CCL2 serumconcentration (data not shown).

Female B6.129S4-Ccl2tm1Rol/J mice were inoculated with theB16-F10_HOMSA_CCL2 tumor cell clone 1A5, as described in above.

Treatment of study groups started 5 days after cell inoculation. Group 1received human IgG vehicle control treatment whereas groups 2 and 3 weretreated i.p. with Mab CKLO2-IgG1 (Fc wild type IgG1) and CKLO2-SG1099((CKLO2 pI-enhanced Fc based on IgG1 with mutations Q311R/P343R (KabatEU numbering)), respectively, at 3.7 mg/kg daily for 9 days. On studyday 14, mice were sacrificed and tumors were explanted. Enzymaticdigestions and cell strainers were used to generate single cellsuspensions from each tumor mass to be analyzed non-pooled by flowcytometry. For the detection of immune cell populations of interestfollowing markers and fluorochromes were used: CD45-BUV395,CD11b-BUV737, F4/80-BV421, CD11c-BV605, Ly6C-AF488, Ly6G-PerCP-Cy5.5,CD206-BV711, CD4-BV510, CD8a-APC-H7, NK1.1-PE-Cy7, CD279-APC andCD274-PE.

Samples were acquired with a BD LSR-Fortessa flow cytometer and analyzeusing the BD Diva Software.

Serum samples were withdrawn on study days 6, 8 11 and 14 to measuretotal and free huCCL2.

The method to detect free CCL2 is described in detail under “Proof ofconcept study of CCL2 sweeping efficiency in cynomolgus monkeys” below.For analysis of free human CCL2 in this study recombinant cynomolgusCCL2 was replaced by recombinant human CCL2 to prepare the calibratorsand QCs.

Total CCL2 serum samples were analyzed with a non-validated, butqualified, specific sandwich ELISA. Briefly, biotinylated anti-CCL2capture antibody (CNTO888 CCL2-0004), blocking buffer, pretreated testsample and detection reagent (digoxigenylated anti-CCL2 antibody(M-1H11-IgG)), were added stepwise to 384-well streptavidin-coatedmicrotiter plate and incubated on a non-vigorous shaker for 1 hour ineach step. To dissociate CCL2-drug complexes in the pre-treatment stepsamples, calibrators or QCs were acidified in pH 5.5 at 37° C. for 10minutes. Acidified samples were added to the SA-MTP. For detection ofimmobilized immune complexes, a polyclonal anti-digoxigenin-PODconjugate was added and the plate was incubated for 60 minutes. Theplate was washed three times after each step to remove unboundsubstances.

ABTS was added to the plate and incubated at room temperature withshaking. Absorption was measured at 405/490 nm wavelength. The humanCCL2 concentrations were calculated based on the response of thecalibration curve using the analytical software XLFit (IDBS).

Depending on the data sets being analyzed statistically a t-test orone-way ANOVA with Tukey's test for multiple comparisons were applied,accordingly.

Results

At end of the study, tumor volumes and tumor weights were significantlyreduced in those mice receiving CKLO2-SG1099 (CKLO2 pI-enhanced Fc)(FIG. 9 ). A closer look at the tumor infiltrate revealed a decreasedtumor infiltrate of monocytic myeloid-derived suppressor cells(M-MDSCs), as expected upon CCL2 blockade (FIG. 9 ).

Furthermore, serum analytics confirmed the efficacy of the therapy: pIoptimization leads indeed to a reduction in the accumulation of totalCCL2 as compared to the IgG1 wild type Fc CKLO2 molecule, whilefree-CCL2 (not bound to antibody) is completely suppressed under limitof detection (FIG. 10 ). Therefore, the present model is suited toinvestigate the effects of CCL2 blockade in a tumor context, usinganti-huCCL2 biparatopic sweeping antibody CKLO2-SG1099 (CKLO2pI-enhanced Fc). In subsequent studies the optimal dose regimen isinvestigated in which CKLO2-SG1099 (CKLO2 pI-enhanced Fc) is given at alower dose once or twice per week and the extension of free-CCL2suppression is monitored over weeks. Furthermore, combination with Tcell activating therapies (i.e. T cell bispecifics, PD-L1 blockade) isalso explored in this model.

In additional analogous experiments other variants like CKLO2-SG1095,CKLO2-GG01, GG02 and GG03/GG04 are analyzed.

Proof of Concept (POC) Study of CCL2 Sweeping Efficiency in CynomolgusMonkeys

Methods. The main objective of this study was to evaluate the extend ofCCL2 suppression and sweeping efficiency of four anti-CCL2 (MCP-1)antibodies in cynomolgus monkeys. The secondary objective was toevaluate the pharmacokinetic (PK) properties of these antibodies. Allantibodies were administered as single IV infusions of 25 mg/kg over aperiod of 30 minutes to 3-4-year-old male animals and total CCL2 andantibody concentrations were measured in serum over 70 days. Theanti-CCL2 antibodies studied comprised of control antibodies (groups 1and 2) as well as antibodies specifically engineered to provide enhancedelimination of CCL2-drug complexes (referred to hereafter as antigensweeping or simply sweeping). Group 1: CNTO888-SG1 (=IgG1 wild type)anti-CCL2 antibody (n=3 animals) as control of maximal total CCL2accumulation; group 2: a biparatopic anti-CCL2 antibody CKLO2-SG1 (IgG1wild type) with pH dependent target binding but no Fc-modifications(n=3); group 3: a biparatopic anti-CCL2 antibody CKLO2-SG1100 with pHdependent target binding and Fc-pI and further modifications (n=4) andgroup 4: biparatopic anti-CCL2 antibody CKLO2-SG1095 with pH dependenttarget binding, Fc-pI and FcγRII and further modifications (n=4).

In this study, the total serum concentrations of the antibodies, thetotal (free and antibody-bound CCL2) and free target were quantified.Furthermore, presence of anti-drug antibodies (ADA) was assessed. Theantibody, total and free CCL2 profiles were analyzed bynon-compartmental analysis using Phoenix 64 (Pharsight/Certara); datawere illustrated using GraphPad Prism v. 6.07 (GraphPad Software).

For the antibodies, serum samples were analyzed using a generic humansandwich ELISA method. The concentrations of total antibody in monkeyserum were measured by ELISA. 2 μg/mL of anti-human kappa chain antibodywas immobilized onto maxisorp 96-well plate overnight before incubatingin blocking buffer for 2 hours at 30° C. Antibody calibration curvesamples, quality control samples and monkey serum samples were incubatedon plate for 1 hour at 30° C. before washing. Next, anti-human IgG-HRPwas added and incubated for 1 hour at 30° C. before washing. ABTSsubstrate was incubated for 10, 20 and 30 minutes before detection withmicroplate reader at 405 nm. The antibody concentrations were calculatedbased on the response of the calibration curve using the analyticalsoftware SOFTmax PRO (Molecular Devices).

Total CCL2 serum samples were analyzed with a non-validated, butqualified, specific sandwich ECL method assay. 3 μg/mL of anti-CCL2antibody (r2F2-SG1) was immobilized onto a MULTI-ARRAY 96-well plate(Meso Scale Discovery) overnight before incubating in blocking bufferfor 2 hours at 30° C. Cynomolgus monkey CCL2 calibration curve samples,quality control samples and diluted cynomolgus monkey serum samples wereincubated with pH5.5 acid buffer for 10 minutes at 37° C. After that,the samples were incubated onto anti-CCL2-immobilized plate for 1 hourat 30° C. before washing. Next, SULFO TAG labelled MCP-1 antibody wasadded and incubated for 1 hour at 30° C. before washing. Read Buffer T(×4) (Meso Scale Discovery) was immediately added to the plate andsignal was detected by SECTOR Imager 2400 (Meso Scale Discovery). Thecynomolgus monkey CCL2 concentrations were calculated based on theresponse of the calibration curve using the analytical software SOFTmaxPRO (Molecular Devices).

Free CCL2 serum samples were analyzed with a non-validated, butqualified, Gyrolab™ immunoassay run on a Gyrolab Xplore. A biotinylatedanti-CCL2 antibody (M-2F6-IgG) was used as capture reagent and fordetection an Alexa647 labeled anti-CCL2 antibody (M-1H11-IgG) wasselected. Both reagents were diluted to 1 μg/mL in PBS, 0.1% Tween, 1%BSA and transferred to a 96-well PCR plate (Fisher Scientific).Cynomolgus monkey CCL2 calibration curve samples, QCs and undilutedserum samples were also transferred to a 96-well PCR plate. Both plateswere loaded into the instrument together with a Gyrolab Bioaffy 200 nldisc (Gyros Protein Technologies AB). A three step assay protocol(200-3W-001) was selected. Briefly the protocol describes the sequentialaddition of capture reagent, sample and detection reagent to designatedstreptavidin columns of the Gyrolab Bio Affy 200 disc. Each reagentreaches the column at the same time after a short spinning step isapplied to the disc. The columns were washed with PBS 0.05% Tween aftereach step and finally laser induced fluorescence values were recordedwithin the instrument. The free cynomolgus monkey CCL2 concentration wascalculated based on the response of the calibration curve using XL Fitsoftware (IDBS).

ADA were analyzed using a method described elsewhere (Stubenrauch etal., 2010). In summary, biotinylated mAb anti-human Fcγ-pan R10Z8E9 wasbound to streptavidin-coated high bind plate at a concentration of 0.5μg/mL and incubated for 1 h. Samples and standards were diluted withassay buffer to 5% cynomolgus monkey serum and added to each well of thecoated plate after washing and incubated for 1 h with shaking. Afterwashing, digoxigenylated anti-cynomolgus(cyno) IgG at 0.1 μg/mL wereadded and incubated for 1 h with shaking. After washing, the polyclonalanti-digoxigenin-HRP conjugate at 25 mU/mL were added and incubated for1 h with shaking. ABTS was added to the plate and incubated for 10minutes at room temperature with shaking. Absorption was measured bymicroplate reader at 405/490 nm wavelength. The ADA concentration wascalculated based on the response of the calibration curve using theanalytical software SOFTmax PRO (Molecular Devices).

Results. The PK behaviour was assessed during the time in which animalswere free of ADA (i.e., before day 14). During this period, the serumconcentration-time profiles of all anti-CCL2 antibodies were similar(see FIG. 11 left panel) and partial average AUC values (AUC_(0-7d))were comparable between the different groups 1490, 1810, 1210 and 1320day. μg/mL for groups 1, 2, 3, and 4 respectively. Similarly, theaverage C_(max) values were comparable with values of 620, 764, 616 and664 μg/mL for the groups 1, 2, 4 and 4, respectively. The extent of ADAdevelopment was highly variable between animals and groups and resultedin highly variable PK profiles beyond day 7 (data not shown). One animalfrom group 2 was ADA-negative throughout the entire observation periodof 70 days (see FIG. 11 right panel). In this animal the clearance,volume of distribution and terminal half-life of anti-CCL2 antibodieswas estimated by non-compartmental analysis at 7.34 mL/(day·kg), 76.2mL/kg and 10.9 days, respectively.

Baseline levels of CCL2 in serum were assessed for each animal beforeantibody treatment started. Basal CCL2 levels ranged from 0.126 to 0.357ng/mL (geometric mean (% CV): 0.199 ng/mL (32.2%, N=14)). As the freeform of CCL2 has a higher elimination rate than the antibody-bound formof CCL2, an increase of total CCL2 serum concentrations followingantibody treatment was expected. This was indeed observed in all groups(FIG. 12 left panel), demonstrating engagement of the target by allantibodies. Under treatment, the C_(max) values of total CCL2 increasedto 824, 575, 106, 32.7 ng/mL for groups 1, 2, 3 and 4, respectively. TheAUC_(0-7d) values were 3060, 2970, 522 and 181 day·ng/mL for groups 1,2, 3 and 4, respectively. During the time that animals were ADAnegative, the molar drug concentrations remained in excess of the totalCCL2 concentrations. The two sweeping anti-CCL2 antibodies (groups 3 and4) showed a considerable reduction in total CCL2 serum concentrationscompared to the conventional antibody (group 1) of approximately 8- and25-fold, respectively based on serum C_(max) values and approximately 6-to 17-fold based on the AUC_(0-7d) values of total CCL2. TheADA-negative animal of Group 2 displayed a sustained target engagement(apparent by the plateau) of total CCL2 concentrations (FIG. 12 rightpanel).

Treatments with all antibodies led to a substantial reduction of freeCCL2 levels in serum (FIG. 13 left panel), however the reduction was inpart only initially. In group 1, all individuals had quantifiable levelsof free CCL2 again after one day. In group 2, all individuals hadquantifiable levels of free CCL2 again after two days. In groups 3 and4, two individuals of each group showed suppression of free CCL2 forseven days. In group 3, two animals with a moderate ADA response,maintaining sufficient antibody concentrations, showed free CCL2suppression below the detection limit for 21 days. In ADA positiveanimals, antibody elimination was significantly increased and as aconsequence of loss of target engagement CCL2 levels returned rapidly totheir original baseline (not shown here).

In additional analogous experiments other variants like CKLO2-SG1099,CKLO2-GG01, GG02 and GG03/GG04 are analyzed.

PK/PD Study of CCL2 Sweeping Efficiency in Cynomolgus Monkeys

Study outline and aims. The PK/PD study was designed based on theresults of the POC study using anti-CCL2 antibody CKL02-SG1095. As thePOC study had demonstrated a high extent of anti-drug antibody (ADA)formation, a Gazyva® (obinituzumab) treatment was included in the PK/PDstudy with the intention to reduce the ADA response. For this purpose,30 mg/kg doses of Gazyva were administered by intravenous infusions fourtime throughout the study: on days −14, −7, 8 and 36. CKL02-SG1095 wasadministered at 2.5, 10 and 25 mg/kg dose levels to four animals (2/2male and female) per dose group as IV infusion over 30 minutes on day 1(groups 1-3). For comparison a conventional anti-CCL2 antibody(CNTO888-IgG1) was administered at 25 mg/kg as IV infusion over 30minutes on day 1 (group 4; same control as group 1 of the POC studydescribed above). Total PK (CKL02-SG1095), total and free CCL2concentrations were assessed until day 99 (i.e., 14 weeks post dose).

The aim of the PK/PD study was to demonstrate a prolonged duration offree CCL2 suppression of CKL02-SG1095 in comparison to a conventionalanti-CCL2 antibody (CNTO888 with wild type IgG1 Fc part) in non-humanprimates.

Methods. Total PK as well as total and free CCL2 were quantified in thisstudy. However, due to the presence of Gazyva in the serum samples somemodifications were made to the total PK assay, the total CCL2 assay andthe ADA assay in comparison to the POC study, which are describedherein. For CNTO888 IgG1 no PK assay was developed.

The concentration of total antibody CKL02-SG1095 in monkey serum wasmeasured by ELISA. For the ELISA biotinylated recombinant human CCL2(Antigen), pre-treated test samples, positive control standards(calibrator) or QCs (quality controls) and digoxigenylated anti-humanIgG (M-1.19.31-IgG) were successively added to a 384 well streptavidincoated microtiter plate (SA-MTP). To dissociate CCL2-drug complexes apre-treatment of test samples was performed at pH 5.5 for 20 minutes.Before addition to the SA-MTP the acidified samples were neutralized.Immobilized immune complexes were detected with a polyclonalanti-digoxigenin-POD conjugate. The plate was washed three times aftereach step to remove unbound substances. ABTS was added to the plate assubstrate and incubated at room temperature. Absorption was measured at405/490 nm wavelength. The antibody concentrations were calculated basedon the response of the calibration curve using the analytical softwareXLFit (IDBS).

Total CCL2 serum samples were analyzed with a non-validated, butqualified, specific sandwich ELISA. Briefly, biotinylated anti-CCL2capture antibody*, pretreated test sample and detection reagent(digoxigenylated anti-CCL2 antibody (1H11-IgG1)), were added stepwise toa 384-well streptavidin-coated microtiter plate and incubated on anon-vigorous shaker for 1 hour for capture and sample step and 50minutes for the detection reagent respectively. To dissociate CCL2-drugcomplexes in the pre-treatment step samples, calibrators or QCs wereacidified in pH 5.5 for 20 minutes. Acidified samples were added to theSA-MTP. For detection of immobilized immune complexes, a polyclonalanti-digoxigenin-POD conjugate was added and the plate was incubated for50 minutes. The plate was washed three times after each step to removeunbound substances.

ABTS was added to the plate and incubated at room temperature withshaking. Absorption was measured at 405/490 nm wavelength. Thecynomolgus monkey CCL2 concentrations were calculated based on theresponse of the calibration curve using the analytical software XLFit(IDBS). *Capture antibody for analysis of Group 1-Group 3: anti-CCL2CNTO8888 IgG1, Capture antibody for analysis of Group 4: anti-CCL2 2F2IgG1.

ADAs were screened with a bridging sandwich ELISA in 384-well plates.Test samples of animals of group 1, 2 and 3, quality control samples andpositive controls were incubated overnight with biotinylated captureantibody CKL02-SG1095 and digoxigenylated detection antibodyCKL02-SG1095 together with two additional anti CCL2 antibodies (2F6-IgG1and 1H11-IgG1) at RT, 500 rpm on a MTP-shaker; these antibodies wereadded to neutralize CCL2. For samples of animals of group 4 biotinlabelled CNTO888-SG1 and digoxigenylated CNTO888-SG1 were usedrespectively. Formed immune complexes were transferred to a Streptavidin(SA)-coated MTP to immobilize the immune complexes via thebiotin-labelled (Bi) capture antibody. Following aspiration of thesupernatant, unbound substances were removed by repeated washing.Detection was accomplished by addition of an anti-digoxigenin POD(p)conjugated antibody and ABTS substrate solution. The color intensity ofthe reaction was photometrically determined (absorption at 405 nm-490 nmreference wavelength). A sample was defined ADA positive if the signalwas found to be above a plate specific cut-point. This cut point wasdefined during assay qualification.

Results. In spite of the Gazyva® (obinituzumab) pre-treatment, 10 out of12 animals of the CKL02-SG1095 treated and 1 out of 4 of the CNTO888treated animals developed ADA with influence on the drug and biomarkerconcentrations. However, the two ADA-negative animals for CKL02-SG1095were in the 25 mg/kg dose group allowing a direct comparison to theADA-negative animals of the CNTO888 group. The PK behaviour was assessedduring the time in which animals were free of ADAs (i.e., before day10). The PK profiles of the three different dose groups for CKL02-SG1095are shown in FIG. 14 , left panel. The partial average AUC values(AUC_(0-7d)) were 229/191 (male/female), 696/813 and 1492/1346 day·μg/mLfor the dose levels 2.5, 10 and 25 mg/kg, respectively. The C_(max)values were 115/122 (male/female), 369/491 and 869/941 μg/mL for thedose levels 2.5, 10 and 25 mg/kg, respectively. At the highest doselevel, these findings are consistent with the POC study. For the twoADA-negative animals the clearance, volume of distribution and terminalhalf-life of CKL02-SG1095 was estimated by non-compartmental analysis at10.5-17.4 mL/(day·kg), 116-118 mL/kg and 5.8-11.6 days, respectively(see FIG. 14 right panel).

As for the POC study describe above, accumulation of total CCL2 wasobserved upon treatment with anti-CCL2 antibodies (see FIG. 15 leftpanel). Baseline levels of CCL2 were assessed on five occasions beforedrug administration (including one occasion before Gazyva®(obinituzumab) treatment); the average CCL2 baseline value was 0.742ng/mL and Gazyva® (obinituzumab) treatment did not affect basal CCL2levels. The extent of total CCL2 accumulation (values in parenthesisindicate the median fold-change from individual baselines) was dose andconstruct dependent. For CKL02-SG1095, total CCL2 levels increased to22.4 (22), 67.2 (105) and 54.9 (76) ng/mL (median of four animals) for2.5, 10 and 25 mg/kg dose levels. For CNTO888 IgG1, total CCL2 levelincreased to 1490 (3160) ng/mL (median of 4 animals). Comparison of theADA-negative animals of groups 3 and 4 showed a considerably lower levelof accumulation for CKL02-SG1095 compared to CNTO888 (FIG. 15 rightpanel).

Treatments of all study groups lead to a substantial, transientreduction of free CCL2 levels in serum (see FIG. 16 left panel;typically, below the limit of detection (0.01 ng/mL). For allADA-positive animals, free CCL2 levels rapidly returned to baselinevalues after ADA developed (consistent with the loss of drug exposureand a rapid target turnover). For ADA-negative animals (2/4 in group 3)and (3/4 in group 4) the duration of free CCL2 suppression could beassessed throughout the study duration. For the conventional antibodyCNTO888 (group 4), the duration of CCL2 suppression was short,presumably due to the extensive accumulation of the total target (FIG.15 ). While the drug concentration was not quantified (no specific assayavailable for CNTO888), the POC study suggests similar PK propertiesbetween CNTO888 and CKL02-SG1095. For the two ADA-negative animals ofgroup 3 on the other hand, long lasting free CCL2 suppression wasobserved. For one animal, free CCL2 levels remained below the limit ofdetection for 29 days (FIG. 16 right panel).

In additional analogous experiments other variants like CKLO2-SG1099,CKLO2-GG01, GG02 and GG03/GG04 are analyzed.

Prevalence Study of CCL2 in Different Tumor Types

The following IHC prevalence study of CCL2 and its receptor CCR2,including macrophages analysis using CD163/CD68 and CD14, was performedon 121 human matched tumor and serum samples of 6 different indications:

pancreatic cancer (PaC)

colorectal cancer (CRC)

breast cancer (BC)

prostate cancer (PrC)

ovarian cancer (OvC)

gastric cancer (GC)

the following questions were addressed:

-   -   do these tumors (over)express CCL2?    -   is the blood CCL2 level elevated in these tumor patients?    -   is there a correlation between blood and tumor CCL2 levels?    -   is there a correlation between tumor CCL2 and infiltrating CCR2⁺        immune cells?    -   is there a correlation between tumor CCL2 and infiltrating        myeloid cells?        Material and Methods

Histopathological scoring was done semiquantitatively. Additionally,automatical multiplex image analysis was used for CD163/CD68 IHC, andtested for CD14 and CCR2 for immune cell quantification, as well as forCCL2 quantification.

The immunohistological investigation was performed on a set of resectionspecimens of 121 human tumors of 6 different indications: 31 Pancreaticcancer (PaC), 30 colorectal cancer (CRC), 30 breast cancer (BC), 29prostate cancer (PrC), 20 ovarian cancer (OvC), and 10 gastric cancer(GC), provided by Indivumed (Hamburg) and Asterand (Royston/Herts, UK).Tumors were fixed in 4% buffered formaldehyde, paraffin-embedded, cut at2.5 μm thickness, and mounted on Superfrost Plus slides. The mousemonoclonal antibody against CCL2 (clone 2D8, Novusbio NBP2-22115) wasused on the Ventana BXT, following a standard staining Protocol (CC1 for32′, concentration of 1 μg/mL in VBX, Optiview DAB detection system).The rabbit monoclonal antibody against CCR2 (E68, Abcam ab32144) wasused on the Ventana Discovery XT, following a standard staining protocol(CC1 for 32′, concentration 0.8 ug/ml in DS2, Omni-UltraMap HRP DABdetection system). The mouse monoclonal antibody against the monocyticmarker CD14 (Cell Marque EPR3653, RTU) was used on the Ventana DiscoveryUltra, following a standard staining protocol (CC1 for 64′,Omni-UltraMap HRP DAB detection system detection system). The doublestaining against macrophages and M2-like TAMs (tumor-associatedmacrophages) CD163/CD68 (DAB CD163 Mouse MRQ-26 Cell Marque RTU/red CD68Mouse PG-M1 Dako) was used on the Ventana BXT, following a standardstaining protocol (CC1 for 32′, CD163 RTU/CD68 concentration 0.6 μg/mlin DS2, Detection with DAB and Red detection systems). All images werescanned using the Ventana iScan HT®. The tissue sections were analyzedsemi-quantitatively.

1. Results

CCL2 and CCR2 Prevalence

All analyzed tumor indications showed some tumors with up-regulation ofCCL2 at variable levels and presence of CCR2 on TAMs at variable amount(Table 6 below). For both, CCL2 and CCR2, the highest expression wasobserved in ovarian carcinoma, followed by PDAC and GC

Tumor Type-Specific Characteristics

Tumors with high activity of CCL2-CCR2, associated with a tumor-growthenhancing immunological status of high MDSC attraction and M2polarization, represent the preferred of tumors for CCL2-blockingtherapy. CCR2 IHC showed a good correlation to MDSCs and M2-likemacrophages confirming its biological role, and demonstrated a higherrelevance as biomarker for this pathway than CCL2 IHC measurement.Concluding from the present study, the following recommendations forCCL2-therapy can be summarized:

-   -   OvC can be recommended for CCL2-targeted therapy because of the        highest CCL2 and CCR2 prevalence and M2 polarization;    -   PDAC can be recommended for CCL2-targeted therapy due to the        highest amount of MDSCs compared to the other analyzed tumor        types, and because CCR2 and M2 were present at considerable high        levels and amounts. CCL2 was high in PDAC as well and showed, in        contrast to the other tumor types, an extraordinary higher        presence in immune cells than in tumor cells. PaC showed a very        good correlation between CCL2/CCR2 and MDSC        attraction/M2-polarization. These results support that in the        analyzed PDACs, the role of CCL2-CCR2 is highly focused on        immune cell attraction.    -   BC, especially TNBC, can be recommended for CCL2-targeted        therapy: BC showed CCL2, CCR2, MDSCs and M2 at considerable high        levels and amounts. Especially TNBC cases were characterized by        higher amounts of M2 and MDSC than non-TNBC, although non-TNBC        showed the highest CCL2 production in tumor cells compared to        other tumor indications.

The following tumor indications seemed to be less dependent from theCCL2-CCR2-axis and, therefore, might be less recommended for CCL2targeted therapy:

-   -   CRC: CCL2 was low compared to the other tumor types, especially        when compared to PaC. Also here, the lowest amount of M2-like        macrophages was measured compared to the other tumor types. CCR2        and MDSCs were present at variable amounts. Interestingly, in        this indication only, a trend of a positive correlation between        CCL2 and CCR2 was detectable. However, the overall findings        support that in CRC, the role of CCL2-CCR2 is focused on tumor        cell survival and not on immune cell attraction.    -   Although high for CCL2, GC was observed to be low for CCR2 and        showed the lowest amount of MDSCs. M2-like macrophages were        present at variable amounts.

CCL2 CCR2 In tumor cells (TC) In immune cells (manual score) In immunecells (manual score) % of (IC) % of mod (manual score) mod % of to hi %of % of to hi positive pos positive Mean positive pos Tumor type: casescases Mean score cases score cases cases Mean Score OvC 100% 42% 1.3100%  0.9 100% 63 1.7 PaC  90%  9% 0.8 97% 2.0  90% 55 1.5 (PDAC (PDAC(PDAC 0.8 and 2.2 and 1.7 and PNET PNET PNET 0.9) 0.6) 0.3) GC  91% 45%1.3 91% 1.9  91% 18 0.8 BC  78% 46% 1.1 61% 0.6  96% 38 1.3 (TNBC (TNBC(TNBC 0.9 0.5 1.2 non- non- non- TNBC TNBC TNBC 1.6 0.9) 1.5) CRC  76%24% 0.8 72% 0.7  96% 39 1.2 PrC  72% 10% 0.7 79% 0.5  72% 10 0.6

-   -   Pr CCR2 was present at variable levels. (M2 and MDSC were not        measured)        Correlation Analyses

The study outcome can be summarized as follows:

-   -   Between tumor CCL2 and CCR2 (IHC), the only positive correlation        was existent in CRC.    -   Serum CCL2 (ELISA) did not correlate with any of the measured        parameters in the tumor, including CCL2, CCR2, macrophages, and        MDSCs. A trend of positive correlation to tumor CCL2 was found        only in PrC, where both methods show very low values.    -   CCR2 expression correlates positively with the presence of        M2-like macrophages and CD14⁺ cells, and negatively with the        M1/M2-ratio, and confirms the biological role of CCR2. The level        of CCR2 correlates better with M2 polarization than with MDSC        attraction.    -   CCL2 showed a trend of positive correlation with MDSC attraction        and M2 polarization. Thus, the CCL2 level alone seemed not to be        the main factor for the presence of MDSCs and M2-like        polarization.

The invention claimed is:
 1. An isolated bispecific antibody comprisinga first antigen-binding site that binds to a first epitope on human CCL2and a second antigen-binding site that binds a second epitope on humanCCL2, wherein A) i) said first antigen-binding site comprises a VHdomain comprising the amino acid sequence of SEQ ID NO:71; and a VLdomain comprising the amino acid sequence of SEQ ID NO:75; and ii) saidsecond antigen-binding site comprises a VH domain comprising the aminoacid sequence of SEQ ID NO:90; and a VL domain comprising the amino acidsequence of SEQ ID NO:93; or B) i) said first antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:71; and a VL domain comprising the amino acid sequence of SEQ IDNO:75; and ii) said second antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:91; and a VL domaincomprising the amino acid sequence of SEQ ID NO:93; or C) i) said firstantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:71; and a VL domain comprising the amino acidsequence of SEQ ID NO:75; and ii) said second antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:90; and a VL domain comprising the amino acid sequence of SEQ IDNO:94; or D) i) said first antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:72; and a VL domaincomprising the amino acid sequence of SEQ ID NO:75; and ii) said secondantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:90; and a VL domain comprising the amino acidsequence of SEQ ID NO:94; or E) i) said first antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:73; and a VL domain comprising the amino acid sequence of SEQ IDNO:75; and ii) said second antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:90; and a VL domaincomprising the amino acid sequence of SEQ ID NO:93; or F) i) said firstantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:73; and a VL domain comprising the amino acidsequence of SEQ ID NO:75; and ii) said second antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:90; and a VL domain comprising the amino acid sequence of SEQ IDNO:94; or G) i) said first antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:73; and a VL domaincomprising the amino acid sequence of SEQ ID NO:75; and ii) said secondantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:92; and a VL domain comprising the amino acidsequence of SEQ ID NO:93; or H) i) said first antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:73; and a VL domain comprising the amino acid sequence of SEQ IDNO:75; and ii) said second antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:91; and a VL domaincomprising the amino acid sequence of SEQ ID NO:93; or I) i) said firstantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:72; and a VL domain comprising the amino acidsequence of SEQ ID NO:75; and ii) said second antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:90; and a VL domain comprising the amino acid sequence of SEQ IDNO:93; or J) i) said first antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:72; and a VL domaincomprising the amino acid sequence of SEQ ID NO:75; and ii) said secondantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:92; and a VL domain comprising the amino acidsequence of SEQ ID NO:93; or K) i) said first antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:72; and a VL domain comprising the amino acid sequence of SEQ IDNO:75; and ii) said second antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:91; and a VL domaincomprising the amino acid sequence of SEQ ID NO:93; or L) i) said firstantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:74; and a VL domain comprising the amino acidsequence of SEQ ID NO:75; and ii) said second antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:90; and a VL domain comprising the amino acid sequence of SEQ IDNO:93; or M) i) said first antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:74; and a VL domaincomprising the amino acid sequence of SEQ ID NO:75; and ii) said secondantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:90; and a VL domain comprising the amino acidsequence of SEQ ID NO:94; or N) i) said first antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:74; and a VL domain comprising the amino acid sequence of SEQ IDNO:75; and ii) said second antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:92; and a VL domaincomprising the amino acid sequence of SEQ ID NO:93; or O) i) said firstantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:74; and a VL domain comprising the amino acidsequence of SEQ ID NO:75; and ii) said second antigen-binding sitecomprises a VH domain comprising the amino acid sequence of SEQ IDNO:91; and a VL domain comprising the amino acid sequence of SEQ IDNO:93; or P) i) said first antigen-binding site comprises a VH domaincomprising the amino acid sequence of SEQ ID NO:71; and a VL domaincomprising the amino acid sequence of SEQ ID NO:75; and ii) said secondantigen-binding site comprises a VH domain comprising the amino acidsequence of SEQ ID NO:92; and a VL domain comprising the amino acidsequence of SEQ ID NO:93.
 2. A composition comprising the bispecificantibody of claim 1, and a carrier.